Articles and methods directed to personalized therapy of cancer

ABSTRACT

Described are methods for providing personalized medicine for the treatment of B cell malignancies including lymphoma. The methods make use of Chimeric Antigen Receptor (CAR) technology.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/753,635, filed Apr. 3, 2020, which is a national stage filing under35 U.S.C. § 371 of international PCT application, PCT/RU2018/000653,filed Oct. 4, 2018, which claims priority to Russian Application No.2017134483, filed Oct. 4, 2017, Russian Application No. 2018112009,filed Apr. 4, 2018, and Russian Application No. 2018134321, filed Oct.1, 2018, the entire contents of each of which is hereby incorporatedherein by reference.

BACKGROUND OF INVENTION

Lymphoma is a cancer in the lymphatic cells of the immune system.Typically, lymphomas present as a solid tumor of lymphoid cells. Thesemalignant cells often originate in lymph nodes, presenting as anenlargement of the node, i.e., a tumor. It can also affect other organsin which case it is referred to as extranodal lymphoma. Extranodal sitesinclude the skin, brain, bowels and bone. Lymphomas are closely relatedto lymphoid leukemias, which also originate in lymphocytes but typicallyinvolve only circulating blood and the bone marrow and do not usuallyform static tumors (Parham, P. The immune system. New York: GarlandScience. p. 414, 2005). Treatment involves chemotherapy and in somecases radiotherapy and/or bone marrow transplantation, and can becurable depending on the histology, type, and stage of the disease. Moreadvanced cases of lymphoma are resistant and, accordingly, noveltreatment approaches are needed.

SUMMARY OF INVENTION

The disclosure provides methods for treatment of B cell malignanciesusing personalized medicine. More particularly, the methods provide forisolating a B cell receptor from a B cell malignancy in a subject,identifying a ligand for the B cell receptor, and then treating thesubject with the B cell receptor ligand coupled to a therapeutic agent,e.g., a CART cell in which the B cell receptor ligand comprises theantigen binding domain.

In some embodiments, the methods of the disclosure use anautocrine-based format to identify B cell receptor ligands specific to atumor. Once a B cell receptor ligand is identified, a patient can betreated with the ligand attached to a therapeutic agent. The wholeprocess, from diagnosis to treatment can be completed in a short periodof time, e.g., within several weeks. As an example, B cell receptorligands may be identified by co-expressing a B cell receptor from atumor and a chimeric antigen receptor (CAR) in a T cell, where theextracellular domain of the CAR comprises a peptide from a library.Activation of the T cell by the CAR indicates that the extracellulardomain of the CAR has bound the B cell receptor and the peptide from thepeptide library is a B cell receptor ligand. Alternatively, contemplatedherein is the use of phage display for identification of the B cellreceptor ligand.

The disclosure also provides methods for treatment of cancer byadministering CAR-expressing T-cells, wherein the CAR comprises anantigen binding domain that specifically binds a cancer-specific antigenin a cancer-specific manner, e.g., a CAR with an antigen binding domaincomprising a B cell receptor ligand as is described herein; and avaccine comprising a polypeptide or a nucleic acid expressing the samecancer-specific antigen, or a cancer-specific fragment thereof, e.g., aB cell receptor or fragment thereof. It has surprisingly been discoveredthat when a CAR specific for a cancer antigen and that same antigen areadministered to a subject, the two have a synergistic effect on areduction in tumor volume.

In one aspect, provided herein are methods of treating lymphoma in asubject. The methods comprise:

identifying a unique B cell receptor expressed in lymphoma cells of thesubject;

expressing the unique B cell receptor in a cell;

contacting the cell with a putative unique B cell receptor ligand from alibrary;

detecting binding of said unique B cell receptor to a putative unique Bcell receptor ligand, thereby identifying a unique B cell receptorligand; and

administering to the subject a therapeutically effective amount of the Bcell receptor ligand coupled to a therapeutic agent.

In some embodiments, the putative unique B cell receptor ligandcomprises a peptide, a cyclopeptide, a peptoid, a cyclopeptoid, apolysaccharide, a lipid, or a small molecule. In some embodiments, theunique B cell receptor and the putative unique B cell receptor ligandare co-expressed in T cells. In some embodiments, the cell comprises aCAR comprising the putative unique B cell receptor ligand.

In some embodiments, the unique B cell receptor is contacted with aputative unique B cell receptor ligand from a library by phage display.In some embodiments, the library comprises a library of putative B cellreceptor ligands linked to a phage. In some embodiments, the unique Bcell receptor is attached to a solid support. In some embodiments,contacting unique B cell receptor with a putative unique B cell receptorligand from a library comprises panning the unique B cell receptorattached to a solid support with the library of putative B cell receptorligands linked to a phage for one or more rounds. In some embodiments,each round of the panning includes negative selection.

In some embodiments, said detection method comprises identifyingactivation of the T cell.

In another aspect, provided herein are methods of treating lymphoma in asubject. The methods comprise:

identifying a unique B cell receptor expressed in lymphoma cells of thesubject;

co-expressing the unique B cell receptor and putative unique B cellreceptor ligand from a library in a cell;

detecting binding of said unique B cell receptor to a putative unique Bcell receptor ligand, thereby identifying a unique B cell receptorligand; and

administering to the subject a therapeutically effective amount of the Bcell receptor ligand coupled to a therapeutic agent.

In some embodiments, the unique B cell receptor and the putative uniqueB cell receptor ligand are co-expressed in T cells.

In some embodiments, the T cell comprises a CAR comprising the putativeunique B cell receptor ligand.

In some embodiments, said detection method comprises identifyingactivation of the T cell.

In some embodiment, the subject is administered the B cell receptor, ora fragment thereof, concomitantly with the therapeutic agent.

In another aspect, provided herein are methods of identifying a B cellreceptor ligand. The methods comprise:

providing to a population of T cells nucleic acid molecules encoding a Bcell receptor and a library of chimeric antigen receptors (CARs),wherein each CAR within the library comprises a distinct putative B cellreceptor ligand domain;

coexpressing the B cell receptor and the library of CARs in T cells;

measuring activation of the T cells, wherein the putative B cellreceptor ligand domain of a CAR from the library of CARs comprises aligand of the B cell receptor if a T cell expressing the B cell receptorand the CAR is activated; and

isolating the nucleic acid molecule encoding the CAR from an activated Tcell; and

sequencing the putative B cell receptor ligand domain of the nucleicacid molecule encoding the CAR from the activated T cell;

thereby identifying a B cell receptor ligand.

In some embodiments, the B cell receptor is from a cancer cell. In someembodiments, the cancer cell is a lymphoma cell. In some embodiments,the lymphoma cell is obtained from a tumor from a patient

In some embodiments, the methods further comprise treating a subjecthaving lymphoma with the B cell receptor ligand wherein the B cellreceptor is expressed in a tumor from the subject; and the B cellreceptor ligand coupled to a therapeutic agent.

In some embodiment, the subject is administered the B cell receptor, ora fragment thereof, concomitantly with the therapeutic agent.

In another aspect, provided herein are methods of treating lymphoma. Themethods comprise:

administering to a subject a therapeutically effective dose of a B cellreceptor ligand coupled to a therapeutic agent,

wherein the B cell receptor ligand comprises a putative B cell receptorligand domain, and wherein a CAR comprising the putative B cell receptorligand domain activates a T cell when co-expressed with the B cellreceptor of the lymphoma cells.

In another aspect, provided herein are methods of treating lymphoma in asubject. The methods comprise:

identifying a unique B cell receptor expressed in lymphoma cells of thesubject;

co-expressing the unique B cell receptor and a chimeric antigen receptor(CAR) from a library of CARs in a T cell, wherein each CAR within thelibrary comprises a distinct putative B cell receptor ligand domain;

identifying a B cell receptor ligand by identifying an activated T cell,wherein the putative B cell receptor ligand domain of the CAR from thelibrary of CARs comprises a ligand of the unique B cell receptor if theT cell expressing the B cell receptor and the CAR is activated; and

administering to the subject a therapeutically effective dose of the Bcell receptor ligand coupled to a therapeutic agent.

In some embodiments, the methods further comprise preparing the B cellreceptor ligand coupled to a therapeutic agent.

In some embodiments, the T cell is activated by autocrine-basedactivation of the CAR.

In some embodiments, identifying a B cell receptor ligand furthercomprises isolating the nucleic acid molecule encoding the CAR from theactivated T cell; and sequencing the putative B cell receptor liganddomain of the nucleic acid molecule encoding the CAR from the activatedT cell.

In some embodiment, the subject is administered the B cell receptor, ora fragment thereof, concomitantly with the therapeutic agent.

In another aspect, provided herein are methods of treating lymphoma in asubject comprising:

identifying a unique B cell receptor expressed in lymphoma cells of thesubject;

co-expressing the unique B cell receptor and a putative unique B cellreceptor ligand from a library in a cell;

identifying said unique B cell receptor ligand by a detection method,wherein a putative unique B cell receptor ligand is a unique B cellreceptor ligand if it interacts with the unique B cell receptor; and

administering to the subject a therapeutically effective amount of the Bcell receptor ligand coupled to a therapeutic agent.

In some embodiments, the unique B cell receptor and a putative unique Bcell receptor ligand are co-expressed in T cells.

In some embodiments, the cell comprises a CAR comprising the putativeunique B cell receptor ligand.

In some embodiments, said detection method comprises identifyingactivation of the T cell.

In some embodiment, the subject is administered the B cell receptor, ora fragment thereof, concomitantly with the therapeutic agent.

In another aspect, provided herein are methods for treating lymphoma ina subject comprising: administering to the subject a therapeuticallyeffective amount of a CART cell expressing a first CAR, wherein:

(i) the first CAR comprises an antigen binding domain that comprises apolypeptide from a cyclopeptide library that binds a unique B cellreceptor expressed in lymphoma cells of the subject,

(ii) the antigen binding domain is identified by

-   -   (a) identifying the unique B cell receptor expressed in lymphoma        cells of the subject;    -   (b) co-expressing the unique B cell receptor and a second CAR        from a library of CARs in a T cell, wherein each CAR within the        library comprises a distinct putative ligand domain that        comprises a polypeptide from a cyclopeptide library; and    -   (c) identifying the antigen binding domain of the first CAR by        identifying an

activated T cell, wherein the putative B cell receptor ligand domain ofthe second CAR from the library of CARs comprises the antigen bindingdomain of the first CAR if the T cell expressing the B cell receptor andthe second CAR is activated; and

(iii) the first CAR has greater specificity and/or activity than acontrol.

In some embodiments, the control comprises a CART cell. In someembodiments, the antigen binding domain of the CAR expressed by the CARTcell binds a ligand other than a B-cell receptor. In some embodiments,the antigen binding domain binds CD-19.

In some embodiments, the first CAR and the second CAR are the same CAR.In some embodiments, the first CAR and the second CAR are differentCARs.

In some embodiments, activity comprises cytotoxicity towards cellsexpressing the unique B cell receptor relative to a control. In someembodiments, cytotoxicity of the CART towards cells expressing theunique B cell receptor is 0%-10% greater than the control, as measuredby % lysis, at an effector:target ratio of 1:1-10:1. In someembodiments, cytotoxicity of the CART towards cells expressing theunique B cell receptor is at least 10% greater than the control, asmeasured by % lysis, at an effector:target ratio of 10:1 or greater.

In some embodiments, the control comprises a CAR comprising an antigenbinding domain that binds a ligand other than the B-cell receptorexpressed on the cells expressing the unique B cell receptor.

In some embodiments, specificity comprises cytotoxicity towards cellsthat do not express the unique B cell receptor. In some embodiments,cytotoxicity of the CART towards cells that do not express the unique Bcell receptor is less than 10%, as measured by % lysis. In someembodiments, cytotoxicity of the CART towards cells that do not expressthe unique B cell receptor is 0-10% less than the cytotoxicity of acontrol that binds a ligand expressed on the cells at an effector:targetratio of less than 10:1. In some embodiments, cytotoxicity of the CARTtowards cells that do not express the unique B cell receptor is at least15% less than the cytotoxicity of a control that binds a ligandexpressed on the cells at an effector:target ratio of 10:1 or greater.

In some embodiment, the subject is administered the B cell receptor, ora fragment thereof, concomitantly with the therapeutic agent.

In another aspect, provided herein are methods for treating lymphoma insubject population comprising:

selecting subjects having lymphoma; and

administering to each subject a therapeutically effective amount of aCART cell expressing a first CAR unique to the B cell receptor expressedon the lymphoma cells on each subject, wherein:

(i) the first CAR comprises an antigen binding domain that comprises apolypeptide from a cyclopeptide library that binds a unique B cellreceptor expressed in lymphoma cells of each subject,

(ii) the antigen binding domain is identified by

-   -   (a) identifying the unique B cell receptor expressed in lymphoma        cells of the subject;    -   (b) co-expressing the unique B cell receptor and a second CAR        from a library of CARs in a T cell, wherein each CAR within the        library comprises a distinct putative ligand domain that        comprises a polypeptide from a cyclopeptide library; and    -   (c) identifying the antigen binding domain of the first CAR by        identifying an activated T cell, wherein the putative B cell        receptor ligand domain of the second CAR from the library of        CARs comprises the antigen binding domain of the first CAR if        the T cell expressing the B cell receptor and the second CAR is        activated; and

(iii) the first CAR has greater specificity and/or activity than acontrol.

In some embodiments, the control comprises a CART cell. In someembodiments, the antigen binding domain of the CAR expressed by the CARTcell binds a ligand other than a B-cell receptor. In some embodiments,the antigen binding domain binds CD-19.

In some embodiments, the first CAR and the second CAR are the same CAR.In some embodiments, the first CAR and the second CAR are differentCARs.

In some embodiments, activity comprises cytotoxicity towards cellsexpressing the unique B cell receptor relative to a control. In someembodiments, cytotoxicity of the CART towards cells expressing theunique B cell receptor is 0%-10% greater than the control, as measuredby % lysis, at an effector:target ratio of 1:1-10:1. In someembodiments, cytotoxicity of the CART towards cells expressing theunique B cell receptor is at least 10% greater than the control, asmeasured by % lysis, at an effector:target ratio of 10:1 or greater.

In some embodiments, the control comprises a CAR comprising an antigenbinding domain that binds a ligand other than the B-cell receptorexpressed on the cells expressing the unique B cell receptor.

In some embodiments, specificity comprises cytotoxicity towards cellsthat do not express the unique B cell receptor. In some embodiments,cytotoxicity of the CART towards cells that do not express the unique Bcell receptor is less than 10%, as measured by % lysis. In someembodiments, cytotoxicity of the CART towards cells that do not expressthe unique B cell receptor is 0-10% less than the cytotoxicity of acontrol that binds a ligand expressed on the cells at an effector:targetratio of less than 10:1. In some embodiments, cytotoxicity of the CARTtowards cells that do not express the unique B cell receptor is at least15% less than the cytotoxicity of a control that binds a ligandexpressed on the cells at an effector:target ratio of 10:1 or greater.

In another aspect, provided herein are methods of of rapidly identifyinga personalized antibody binding ligand specific for a B cell lymphoma,e.g., a B cell receptor ligand, comprising:

identifying a B cell receptor from a B cell lymphoma cell,

providing to a population of T cells nucleic acid molecules encoding theB cell receptor and a library of chimeric antigen receptors (CARs),wherein each CAR within the library comprises a distinct putative B cellreceptor ligand domain;

coexpressing the B cell receptor and the library of CARs in T cells;

measuring activation of the T cells, wherein the putative B cellreceptor ligand domain of a CAR from the library of CARs comprises aligand of the B cell receptor if a T cell expressing the B cell receptorand the CAR is activated; and

isolating the nucleic acid molecule encoding the CAR from an activated Tcell; and

sequencing the putative B cell receptor ligand domain of the nucleicacid molecule encoding the CAR from the activated T cell;

thereby identifying a B cell receptor ligand.

In some embodiments, the B cell receptor ligand is identified within 4weeks, within 3 weeks, within 2 weeks, or within 1 week. In someembodiments, the B cell receptor ligand is identified within 3 weeks.

In some embodiments, the B cell lymphoma cell is obtained from a tumorfrom a patient.

In some embodiments, the putative B cell receptor ligand domaincomprises a polypeptide of 30 amino acids or less. In some embodiments,the putative B cell receptor ligand domain comprises a polypeptide froma cyclopeptide library. In some embodiments, the putative B cellreceptor ligand domain further comprises an Fc region.

In some embodiments, T cell activation is measured by an increase inexpression of CD69 or CD25. In some embodiments, T cell activation ismeasured by an increase in expression of a fluorescent protein reportergene under the control of Jun, NF-κB and/or Rel.

In some embodiments, the methods further comprise treating a subjecthaving lymphoma with the B cell receptor ligand, wherein the B cellreceptor ligand coupled to a therapeutic agent.

In another aspect, provided herein is a chimeric antigen receptor (CAR)comprising: a putative B cell receptor ligand domain that comprises apolypeptide from a cyclopeptide library;

a transmembrane domain; and

an intracellular region.

In some embodiments, the CAR activates a T cell when co-expressed with aB cell receptor, wherein a B cell receptor ligand of the B cell receptorcomprises the putative B cell receptor ligand domain. In someembodiments, the B cell receptor ligand comprises the amino acidsequence of any of SEQ ID NOs: 1-3.

In another aspect, provided herein is a method of treating lymphoma in asubject comprising:

identifying a unique B cell receptor expressed in lymphoma cells of thesubject;

contacting the unique B cell receptor with a phage display library,wherein the phage display library comprises a library of putative uniqueB cell receptor ligands linked to phages;

detecting binding of said unique B cell receptor to a putative unique Bcell receptor ligand, thereby identifying a unique B cell receptorligand; and

administering to the subject a therapeutically effective amount of the Bcell receptor ligand coupled to a therapeutic agent.

In some embodiments, the putative unique B cell receptor ligandcomprises a peptide, a cyclopeptide, a peptoid, a cyclopeptoid, apolysaccharide, a lipid, or a small molecule.

In some embodiments, the unique B cell receptor is attached to a solidsupport.

In some embodiments, contacting unique B cell receptor with a putativeunique B cell receptor ligand from a library comprises panning theunique B cell receptor attached to a solid support with the library ofputative B cell receptor ligands linked to a phage for one or morerounds. In some embodiments, each round of the panning includes negativeselection.

In some embodiments, the subject is determined to have lymphoma.

In some embodiments, the subject is determined to have one or moresingle-nucleotide polymorphisms (SNPs) associated with lymphoma.

In some embodiments, identifying a unique B cell receptor comprises:

obtaining cells from a biopsy;

extracting RNA from the cells;

synthesizing cDNA from the extracted RNA; and

sequencing the cDNA. In some embodiments, identifying a unique B cellreceptor comprises cloning and sequencing circulating cell free DNA.

In some embodiments, the method is performed in 3 weeks or less.

In some embodiments, the therapeutic agent comprises a radioactiveisotope.

In some embodiments, the B cell receptor ligand coupled to a therapeuticagent comprises a therapeutic CAR. In some embodiments, the therapeuticagent comprises a chemotherapy. In some embodiments, the therapeuticagent comprises an immunotherapy.

In another aspect, provided herein is a method of treating cancer in asubject. In some embodiments, the method comprises concomitantlyadministering: CAR-expressing T-cells, wherein the CAR comprises anantigen binding domain that specifically binds a cancer-specific antigenin a cancer-specific manner; and a vaccine comprising a polypeptide or anucleic acid expressing the cancer-specific antigen, or acancer-specific fragment thereof.

In some embodiments, the cancer-specific antigen is a B-cell receptor.In some embodiments, the cancer is a lymphoma. In some embodiments, thepolypeptide or nucleic acid comprises a heavy or light chain variableregion, or fragment thereof.

In some embodiments, the cancer-specific antigen is expressed in thecancer and comprises a somatic mutation. In some embodiments, thenon-cancerous cells of the subject do not have the somatic mutation. Insome embodiments, the mutation is a point mutation, a splice-sitemutation, a frameshift mutation, a read-through mutation, or agene-fusion mutation. In some embodiments, the somatic mutationcomprises a mutation in EGFRvIII, PSCA, BCMA, CD30, CEA, CD22, L1CAM,ROR1, ErbB, CD123, IL13Ra2, Mesothelin, FRα, VEGFR, c-Met, 5T4, CD44v6,B7-H4, CD133, CD138, CD33, CD28, GPC3, EphA2, CD19, ACVR2B, anaplasticlymphoma kinase (ALK), MYCN, BCR, HER2, NY-ESO1, MUC1, or MUC16. In someembodiments, the cancer comprises a tumor. In some embodiments, thepolypeptide or nucleic acid comprises the somatic mutation.

In some embodiments, the concomitant administration occurs at least twotimes, at least three times, at least four times, at least five times,at least six times, at least seven times, at least eight times, at leastnine times, or at least ten times in the subject. In some embodiments,the CAR-expressing T-cells are administered before the vaccine. In someembodiments, the CAR-expressing T-cells are administered after thevaccine.

In some embodiments, the method further comprises identifying thecancer-specific antigen in the subject. In some embodiments, identifyingthe cancer-specific antigen comprises: (i) obtaining cancerous cellsfrom a subject; (ii) extracting DNA from the cells; and (iii) sequencingthe DNA. In some embodiments, identifying the cancer-specific antigenfurther comprises comparing the DNA sequence obtained from the cancerouscells to a DNA sequence of the same gene obtained from non-cancerouscells. In some embodiments, the DNA is isolated from tumor cells. Insome embodiments, the cancer-specific antigen comprises isolating andsequencing circulating cell free DNA of the subject. In someembodiments, identifying the cancer-specific antigen comprises: (i)obtaining cancerous cells from a subject; (ii) extracting RNA from thecells; (iii) synthesizing cDNA from the extracted RNA; and (iv)sequencing the cDNA. In some embodiments, identifying thecancer-specific antigen further comprises comparing the cDNA sequenceobtained from the cancerous cells to a cDNA sequence of the same geneobtained from non-cancerous cells.

In some embodiments, the vaccine comprises two or more polypeptideshaving overlapping sequences, each expressing a fragment of thecancer-specific antigen.

In some embodiments, the method further comprises providingCAR-expressing T-cells by: (i) identifying an antigen binding domainthat specifically binds the cancer-specific antigen in a cancer-specificmanner; and (ii) expressing a CAR comprising the antigen binding domainin T-cells.

In some embodiments, the polypeptide is conjugated to KLH.

In some embodiments the vaccine is administered by intravenous,intraperitoneal, transmucosal, oral, subcutaneous, pulmonary,intranasal, intradermal or intramuscular administration. In someembodiments the vaccine is administered intratumorally.

In some embodiments the CAR-expressing T-cells are administered byintravenous administration.

In some embodiments, the method further comprises administering a TLR9agonist. In some embodiments, the cancer-specific antigen is OX40.

In another aspect, provided herein is a composition for treating cancerin a subject comprising: CAR-expressing T-cells, wherein the CARcomprises an antigen binding domain that specifically binds acancer-specific antigen in a cancer-specific manner; and a polypeptideor a nucleic acid expressing the cancer-specific antigen, or acancer-specific fragment thereof.

In some embodiments, the cancer-specific antigen is a B-cell receptor.In some embodiments, the polypeptide or nucleic acid comprises a heavyor light chain variable region, or fragment thereof.

In some embodiments, the cancer-specific antigen is expressed in thecancer and comprises a somatic mutation.

In some embodiments, the non-cancerous cells of the subject do not havethe somatic mutation. In some embodiments, the mutation is a pointmutation, a splice-site mutation, a frameshift mutation, a read-throughmutation, or a gene-fusion mutation. In some embodiments, the somaticmutation comprises a mutation in EGFRvIII, PSCA, BCMA, CD30, CEA, CD22,L1CAM, ROR1, ErbB, CD123, IL13Ra2, Mesothelin, FRα, VEGFR, c-Met, 5T4,CD44v6, B7-H4, CD133, CD138, CD33, CD28, GPC3, EphA2, CD19, ACVR2B,anaplastic lymphoma kinase (ALK), MYCN, BCR, HER2, NY-ESO1, MUC1, orMUC16. In some embodiments, the polypeptide or nucleic acid comprisesthe somatic mutation.

In some embodiments, the vaccine comprises two or more polypeptideshaving overlapping sequences, each expressing a fragment of thecancer-specific antigen.

In some embodiments, the polypeptide is conjugated to KLH.

In some embodiments, the method further comprises administering a TLR9agonist. In some embodiments, the cancer-specific antigen is OX40.

In some embodiments, the CAR, e.g., a CAR described herein, comprises atransmembrane domain. In some embodiments, the transmembrane domaincomprises alpha, beta or zeta chain of the T-cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80,CD86, CD134, CD137 and/or CD154.

In some embodiments, the CAR, e.g., a CAR described herein, comprises anintracellular region. In some embodiments, the intracellular regioncomprises a MHC class I molecule, a TNF receptor protein, anImmunoglobulin-like protein, a cytokine receptor, an integrin, asignaling lymphocytic activation molecule (SLAM protein), an activatingNK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27,CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD25/CD18), 4-29B (CD137), B7-H3,CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD423, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD129, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 304), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD123, and/or a ligand that specifically binds withCD83.

In some embodiments, the CAR, e.g., a CAR described herein, comprises ahinge domain.

In some embodiments, the therapeutic agent comprises a radioactiveisotope. In some embodiments, the B cell receptor ligand coupled to atherapeutic agent comprises a therapeutic CAR. In some embodiments, thetherapeutic agent comprises a chemotherapy. In some embodiments, thetherapeutic agent comprises an immunotherapy.

In some embodiments, identifying a unique B cell receptor comprises:obtaining cells from a biopsy; extracting RNA from the cells;synthesizing cDNA from the extracted RNA; and sequencing the cDNA. Insome embodiments, identifying a unique B cell receptor comprises cloningand sequencing circulating cell free DNA.

In some embodiments, the putative B cell receptor ligand domaincomprises a polypeptide of 30 amino acids or less. In some embodiments,the putative B cell receptor ligand domain comprises a polypeptide froma cyclopeptide library. In some embodiments, the putative B cellreceptor ligand domain further comprises an Fc region.

In some embodiments, T cell activation is measured by an increase inexpression of CD69 or CD25. In some embodiments, T cell activation ismeasured by an increase in expression of a fluorescent protein reportergene under the control of Jun, NF-κB and/or Rel.

In some embodiments, the method is performed in 3 weeks or less.

In some embodiments, the subject is determined to have lymphoma. In someembodiments, the subject is determined to have one or moresingle-nucleotide polymorphisms (SNPs) associated with lymphoma.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein. In the figures:

FIG. 1 is a schematic diagram showing the workflow for selection ofligands for the personalized follicular lymphoma CAR-T therapy. A lymphnode biopsy sample from a patient with Follicular lymphoma is isolatedand the collected tumor cells are used for identification of themalignant BCR genes after which they are reconstituted as a membranebound BCR using PDGFR as a membrane anchor. The reconstituted malignantBCR, co-expressed with the cyclopeptide-CAR library on the surface ofthe Jurkat cell line are used as a reporter-cell system for selection ofthe tumor cell targeting ligand. Following several rounds of panning,the selected peptide ligands fused to the chimeric antigen receptor aresequenced and may be immediately used for generation of the therapeuticT lymphocytes modified by tumor-specific CAR. The sequences top tobottom correspond to SEQ ID NOs: 31 and 32.

FIGS. 2A-2C shows autocrine-based selection of malignant FL-BCR ligands.FIG. 2A shows the reporter system format. FIG. 2B is flow cytometry datashowing verification of the reporter cell assay by Myc-CAR/anti-Mycantibody pair interaction. FIG. 2C shows that patient BCR-specificpeptides on CAR activate reporter Jurkat cells transduced by membranetethered follicular lymphoma BCRs.

FIGS. 3A-3D are graphs showing the selected peptide ligands specificallyinteract with the FL-BCRs and redirects CTLs to kill tumor cells. FIG.3A is a series of histograms showing SPR analysis of the interaction ofthe selected cyclopeptides CILDLPKFC (FL1) (SEQ ID NO: 1), CMPHWQNHC(FL2) (SEQ ID NO: 2), and CTTDQARKC (FL3) (SEQ ID NO: 3) and themalignant BCR. Surface staining of Raji cells transduced with lymphomaBCR scFv by synthetic biotinylated peptides and antibody against IgG Fc.For IgG Fc staining, same Raji cell population flow cytometry result wasused as control in the three histograms. FIG. 3B is a series of graphsshowing % cell lysis. FL-CARTs were co-cultured with Raji cellstransduced with different lymphoma BCRs. Mock transduced T cells andCD19-CART was used as a comparison. Cytotoxicity was determined bymeasuring lactate dehydrogenase release after 6 hours. FIG. 3C showscells from the patient's biopsy or control B-cells were stained with thesynthetic biotinylated FL1 peptide. The B-cell population was identifiedby B220 specific antibody and the FL1 peptide was labeled with biotinand detected with FITC labeled streptavidin. FIG. 3D is a graph showinglysis of B cells derived from the lymphoma biopsy sample by FL1-CARTcompared to Myc-CART and Mock transduced T cells.

FIGS. 4A-4F show CTLs re-directed by FL1-CAR suppress lymphomagenesis invivo. FIG. 4A is a schematic diagram showing experimental designindicating the engraftment of NOD SCID mice with 5×10⁶ Raji-FL1 cells.At day 15, animals (12 per group) were randomized according to the tumorvolume and received i.v. 3×10⁶ FL1-CART, CD19-CAR or Myc-CART per mouseat day 17. FIG. 4B is a series of graphs showing transduction efficacyof activated, CD3/CD28 bead-expanded human CD8⁺ T-cells with lentiviralbased vectors expressing FL1-CAR, Myc-CAR and CD19-CAR constructs. Cellswere stained with IgG1 specific antibody or protein L. FIG. 4C is agraph showing survival of Raji-FL xenografted mice treated on day 17after tumor injection with 3×10⁶ CTLs (n=12 mice per group). Overallsurvival curves were plotted using the Kaplan-Meier method and comparedusing the log-rank (Mantel-Cox) test (*p<0.01). FIG. 4D is a graphshowing a tumor growth curve in groups of mice (n=12) treated by 3×10⁶of FL1-CART, CD19-CART or Myc-CART administered i.v. on day 17 afterinjection of Raji-FL1. Absolute counts of adoptively transferredmodified T cells were monitored in blood obtained from retro-orbitalpuncture using flow cytometry analysis with a CD3⁺ specific antibody(insert). FIG. 4E shows flow cytometry analysis of the phenotype ofFL1-CART cells prior to injection and on day 21 following the injection.FIG. 4F is a graph showing relative percentages of naïve, central memoryand effector memory CART on day 21 following the injection.

FIGS. 5A-5C illustrate the structure of the reconstituted malignant BCRand combinatorial cyclopeptide library. FIG. 5A shows amino acidsequences of the combinatorial cyclopeptide library fused with chimericantigen receptors signaling domains. The sequence corresponds to SEQ IDNO: 33. FIG. 5B shows reconstituted malignant BCR fused with the IgG1 Fchinge and membrane-spanning PDGFR domain. The sequence corresponds toSEQ ID NO: 34. FIG. 5C shows a schematic representation of secretedmolecules.

FIG. 6 shows that FL-CARTs do not eliminate Raji cells without exogenouslymphoma BCR. Only CD-19 CART showed killing activity on regular Rajicells. Minimum unspecific lysis was observed when FL1-CAR, FL2-CAR andFL3-CAR T cells were incubated with Raji cells. Cytotoxicity wasdetermined by measuring lactate dehydrogenase release after 6 hours.

FIGS. 7A-7C shows that CTLs redirected by FL1-CAR infiltrate solidtumors and prevent xenograft metastasis. FIG. 7A shows bioluminescentimaging of organ-specific metastasis of Raji-FL1 cells (green, indicatedby arrows) on day 35 after tumor implantation in mice treated byCD19-CART, FL1-CART and Myc-CART. For the Raji-FL1 cells detection micereceived i.p. injection of the D-luciferine. FIG. 7B showshistopathological changes analysis in tumors from CD19-CART, FL1-CART orMyc-CART treated animals. For identification of the histopathologicalchanges tumors were stained with Hematoxylin-Eosin. Lymphoma B cellswith basophilic cytoplasm and high mitotic rate are indicated as blackarrows, right panel. Macrophages containing cellular debris giving thecharacteristic “starry sky” appearance are indicated by red arrows,right panel. Cells thought to be in the state of apoptosis are indicatedby arrows, left panel. FIG. 7C shows immunohistochemical analysis ofCD19-CART, FL1-CART or Myc-CART infiltration into the tumor (blackarrows). The human CD8-specific antibodies were used for CART staining.

FIGS. 8A-8E show that malignant B cell receptor recognizes self-antigenmyoferlin. FIG. 8A shows a schematic representation of myoferlin-drivenautoreactive lymphomagenesis. FIG. 8B shows PCR analysis of bcl-2rearrangement in FL patient 1 biopsy sample. Staining of HEp-2 cells(FIG. 8C) and myoferlin-expressing HEK293T cells (FIG. 8D) with solublemalignant BCR is shown. Shown in FIG. 8E is an alignment of the aminoacid sequences of the identified malignant-specific peptide FL1 with theprotein Myoferlin and surface proteins from Streptococcus mitis andPneumocytis jirovecii. The sequences from top to bottom correspond toSEQ ID NOs: 1, 35, 36, and 37.

FIG. 9 shows percentages of hCD45+ lymphocytes, CD3+ T cells and CD19+ Bcells in the lymphoid gate of PBMC at different time points followingtransplant.

FIG. 10 shows percentages of CD4+ and CD8+human T cell subsets in thePBMC at different time points following transplant.

FIG. 11 shows levels of human IgM and IgG in humanized mice plasma atdifferent time points following transplant.

FIG. 12 shows tumor growth kinetics in experimental groups.

FIG. 13 shows quantity of CAR T cells on day 38.

FIG. 14 shows levels of hCD45+ lymphocytes, CD3+ T cells and CD19+ Bcells in the lymphoid gate in PBMC.

FIG. 15 shows percentages of CD4+ and CD8+ human T cell subsets in thePBMC.

FIG. 16 shows levels of human IgM and IgG in mice plasma at differenttime points following transplant.

FIG. 17 shows the CAR T lentiviral vector.

FIG. 18 shows tumor growth kinetics in experimental groups.

FIG. 19 shows NNK coding moiety flanked by Cysteines used in the PhageDisplay Cyclopeptide Library Kit used in Example 3. The sequences fromtop to bottom correspond to SEQ ID NOs: 38, 39, and 40.

FIG. 20 shows ELISA results for the binding of phages resulting fromI-III rounds of panning as described in Example 3 against the BCR ofpatient FL1 with the BCR of patients FL1 and FL5. Phage concentrationsare, from left to right, 5, 2.5, 1.25, 0.63, and 0.31 mk/well for eachround of panning for each antibody shown.

DETAILED DESCRIPTION OF INVENTION

The disclosure provides methods for treatment of B cell malignanciesusing personalized medicine. More particularly, the methods provide forisolating a B cell receptor from a B cell malignancy in a subject,identifying a ligand for the B cell receptor, and then treating thesubject with the B cell receptor ligand coupled to a therapeutic agent,e.g., a CART cell in which the B cell receptor ligand comprises theantigen binding domain. In some embodiments, the methods of thedisclosure use an autocrine-based format to identify B cell receptorligands specific to a tumor. By co-expressing a B cell receptor and alibrary of putative B cell receptor ligands, a B cell receptor ligandcan be identified by its binding to the B cell receptor. Alternatively,the B cell receptor ligand can be identified by phage display. The Bcell receptor ligand can be an effective therapeutic when coupled to atherapeutic agent because it can target the therapeutic agent to the Bcell malignancy by binding the B cell receptor. The methods describedherein are particularly useful for treating B cell malignancies becauseB cell tumors are clonal populations having B cell receptors that arepresent in all of the cells of the tumor and only in the cells of thetumor. This allows for the identification of a personalized therapeutictarget with no or very little off target effects.

In some embodiments, the methods described herein utilize autocrinesignaling. As such, the methods described herein make use of autocrinesignaling to identify novel therapeutics for treating B cellmalignancies. As is used herein, “autocrine signaling” refers to a formof cell signaling in which a cell secretes a hormone or chemicalmessenger, e.g., an antigen, that binds to autocrine receptors, e.g., Bcell receptors, on that same cell, leading to changes in the cell.

As an example, B cell receptor ligands may be identified byco-expressing a B cell receptor from a tumor and a CAR in a T cell,where the extracellular domain of the CAR comprises a peptide from acombinatorial peptide library. Activation of the T cell by the CARindicates that the extracellular domain of the CAR has bound the B cellreceptor and the peptide from the peptide library is a B cell ligand.

Once a B cell receptor ligand is identified, a patient can be treatedwith the ligand attached to a therapeutic agent. Therapeutic agents cancomprise chemotherapeutic drugs, immunotherapy, or radioactive isotopes.A CAR comprising the B cell receptor ligand can comprise a therapeuticagent. The CAR can be the same CAR used to identify the B cell receptorligand, allowing for particularly fast identification of a personalizedtherapeutic target and synthesis of personalized medicine.

The whole process, from diagnosis to treatment can be completed in ashort period of time, e.g., within several weeks.

The disclosure also provides methods for treatment of cancer byadministering CAR-expressing T-cells, wherein the CAR comprises anantigen binding domain that specifically binds a cancer-specific antigenin a cancer-specific manner; and a vaccine comprising a polypeptide or anucleic acid expressing the same cancer-specific antigen, or acancer-specific fragment thereof. It has surprisingly been discoveredthat when a CAR specific for a cancer antigen and that same antigen areadministered to a subject, the two have a synergistic effect on areduction in tumor volume.

In some embodiments, the CAR-expressing T cells comprise the CAR withthe putative B cell receptor ligand, and the vaccine comprises afragment or all of the B cell receptor. In some embodiments, theCAR-expressing T cells comprise an antibody fragment to an antigen thatis specific to cancer cells and the vaccine comprises a fragment or allof that same antigen.

B Cell Receptors

The B-cell receptor or BCR is a transmembrane receptor protein locatedon the outer surface of B cells. The receptor's binding moiety iscomposed of a membrane-bound antibody that, like all antibodies, has aunique and randomly determined antigen-binding site generated by V(D)Jrecombination. When a B cell is activated by its first encounter with anantigen that binds to its receptor (its “cognate antigen”), the cellproliferates and differentiates to generate a population ofantibody-secreting plasma B cells and memory B cells.

The BCR complexes with CD79, a transmembrane protein, and generates asignal following recognition of antigen by the BCR. CD79 is composed oftwo distinct chains, CD79A and CD79B, which form a heterodimer on thesurface of a B cell stabilized by disulfide bonding. CD79a and CD79b areboth members of the immunoglobulin superfamily. Both CD79 chains containan immunoreceptor tyrosine-based activation motif (ITAM) in theirintracellular tails that they use to propagate a signal in a B cell, ina similar manner to CD3-generated signal tranduction observed during Tcell receptor activation on T cells.

As used herein, the term “antibody” refers to a protein that includes atleast one immunoglobulin variable domain or immunoglobulin variabledomain sequence. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. An antibody can have the structural features ofIgA, IgG, IgE, IgD, IgM (as well as subtypes thereof).

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917, see also www.hgmp.mrc.ac.uk).Kabat definitions are used herein. Each VH and VL is typically composedof three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The VH or VL chain of the antibody can further include a heavy or lightchain constant region, to thereby form a heavy or light immunoglobulinchain, respectively. In one embodiment, the antibody is a tetramer oftwo heavy immunoglobulin chains and two light immunoglobulin chains,wherein the heavy and light immunoglobulin chains are inter-connectedby, e.g., disulfide bonds. In IgGs, the heavy chain constant regionincludes three immunoglobulin domains, CH1, CH2 and CH3.

B-cell malignancies represent a diverse collection of diseases,including most non-Hodgkin's lymphomas (NHL), some leukemias, andmyelomas. Examples include chronic lymphocytic leukemia, follicularlymphoma, mantle cell lymphoma and diffuse large B-cell lymphoma. B cellmalignancies can be characterized as indolent or aggressive. Indolentmalignancies, such as follicular lymphoma, small lymphocytic lymphomaand marginal zone lymphoma, are characterized by slow growth and a highinitial response rate, followed by a relapsing and progressive diseasecourse. Aggressive lymphomas, such as diffuse large B-cell lymphoma,mantle cell lymphoma and Burkitt's lymphoma, are characterized by rapidgrowth and lower initial response rates, with shorter overall survival(OS).

B cell malignancies are characterized in that they are clonalpopulations of B cells. Since they are clonal populations of B cells,each cancerous cell in the population of cancer cells, e.g., a tumor,has the same B cell receptor. As such, B cell receptors on cancerouscells are tumor specific antigens that can be targeted by the ligand (or“antigen”) of the BCR. Accordingly, disclosed herein are methods foridentifying BCR ligands. Once identified,

BCR ligands can be used, for example, as a cancer treatment. Therapeuticagents can be targeted to cancer cells via the interaction between theBCR and the BCR ligand.

In some embodiments, the methods described herein comprise identifyingor providing a B cell receptor, e.g., expressed in cancer cells. In someembodiments, identifying or providing a B cell receptor comprisesacquiring a sample from a subject. In some embodiments, the sample is afluid sample, e.g., blood. In some embodiments, the sample is a tissuesample. In some embodiments, the sample comprises a, e.g., a tumorsample or a biopsy. In some embodiments, the biopsy is a lymph nodebiopsy.

In some embodiments, the sample is from a subject having or suspected ofhaving cancer. In some embodiments, the cancer is a B cell malignancy.In some embodiments, the cancer is a lymphoma. In some embodiments, thecancer is selected from diffuse large B-cell lymphoma (DLBCL),follicular lymphoma, marginal zone B-cell lymphoma (MZL) ormucosa-associated lymphatic tissue lymphoma (MALT), chronic lymphocyticleukemia (CLL), mantle cell lymphoma (MCL), Burkitt's lymphoma,lymphoplasmacytic lymphoma, nodal marginal zone B cell lymphoma (NMZL),splenic marginal zone lymphoma (SMZL), intravascular large B-celllymphoma, primary effusion lymphoma, lymphomatoid granulomatosis,primary central nervous system lymphoma, ALK-positive large B-celllymphoma, plasmablastic lymphoma, large B-cell lymphoma arising inHHV8-associated multicentric Castleman's disease, and B-cell lymphoma.

In some embodiments, the subject is determined to have any of thecancers described herein. In some embodiments, the subject is determinedto have a B cell malignancy. In some embodiments, the subject isdetermined to have lymphoma. In some embodiments, the subject isdetermined to have one or more single-nucleotide polymorphismsassociated with cancer, e.g., a B cell malignancy and/or lymphoma. Asused herein “single-nucleotide polymorphism” (SNP) refers to a DNAsequence variation occurring when a single nucleotide—A, T, C or G—inthe genome (or other shared sequence) differs between members of abiological species or paired chromosomes in an individual.

In some embodiments, identifying or providing a B cell receptorcomprises extracting RNA out of the cells of the sample. Methods forextracting RNA out of cells are well known to those of skill in the artand include, for example, phenol/chlorophorm based extraction methods,or the use of the RNAeasy Kit™ (Qiagen).

In some embodiments, identifying or providing a B cell receptorcomprises synthesizing cDNA out of extracted RNA. Methods for producingcDNA are well known to those of skill in the art and comprises theformation of cDNA from mRNA by reverse transcriptase.

In some embodiments, identifying or providing a B cell receptorcomprises sequencing the cDNA. The type of sequencing performed can be,for example, pyrosequencing, single-molecule real-time sequencing, iontorrent sequencing, sequencing by synthesis, sequencing by ligation(SOLiD™), and chain termination sequencing (e.g., Sanger sequencing).Sequencing methods are known in the art and commercially available (see,e.g., Ronaghi et al.; Uhlén, M; Nyren, P (1998). “A sequencing methodbased on real-time pyrophosphate”. Science 281 (5375): 363; and Ronaghiet al.; Karamohamed, S; Pettersson, B; Uhlén, M; Nyren, P (1996).“Real-time DNA sequencing using detection of pyrophosphate release”.Analytical Biochemistry 242 (1): 84-9; and services and productsavailable from Roche (454 platform), Illumina (HiSeq and MiSeq systems),Pacific Biosciences (PACBIO RS II), Life Technologies (Ion Proton™systems and SOLiD™ systems)).

In some embodiments, the B cell receptor is cloned into an expressionvector for expressing the B cell receptor in T cells using methodsdescribed herein.

In some embodiments, the B cell receptor is cloned into an scFv formatusing a vector, e.g., a pComb3X vector.

In some embodiments, the scFv form of the B cell receptor is cloned intoa vector for expressing the antibody molecules as dimers with thevariable region in the plasma membrane with their binding sites facingthe solvent. In some embodiments, the scFv form of the B cell receptoris cloned into a vector containing a linker. In some embodiments, thelinker is a a flexible linker to a membrane-spanning domain of theplatelet-derived growth factor receptor. In some embodiments, the vectorfurther comprises a constant domain of antibody, e.g., Fc, e.g., IgG1Fc.

Identifying B-Cell Receptor Ligands

In some embodiments, the methods described herein comprise identifying aB cell receptor ligand. Once the B cell receptor is identified, theligand of the B cell receptor is identified by contacting the B cellreceptor with putative B cell receptor ligands, e.g., a library ofputative B cell receptor ligands.

In some embodiments, the methods described herein provide forco-expressing B cell receptors and a library of putative B cell receptorligands in cells, e.g., T cells, and detecting binding of the B cellreceptor to a putative B cell receptor ligand, thereby identifying aunique B cell receptor ligand.

In some embodiments, detecting binding comprises measuring the level ofB cell receptor signaling. When the B cell receptor and a putative Bcell receptor ligand are both expressed, e.g., in a B cell, if theputative B cell receptor ligand is a ligand of the B cell receptor, thebinding of the B cell receptor will initiate a signaling cascade. Itsome embodiments, detecting binding comprises measuring the expressionof genes regulated by BCR signaling.

In some embodiments, the methods described herein provide forco-expressing B cell receptors and a library of CARs comprising putativeB cell receptor ligand domains in T cells and detecting binding of the Bcell receptor to a putative B cell receptor ligand by identifyingactivation of the T cell by the CAR, thereby identifying a unique B cellreceptor ligand.

In some embodiments, T cells are transduced or transfected with nucleicacids encoding B cell receptors and CARs and T cell activation ismeasured after a period of time. In some embodiments, T cell activationis measured 2, 4, 6, 8, 12, 16, or 20 hours, or 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, or 5 days after transduction or transfection, e.g., 2 days aftertransduction or transfection.

In some embodiments, co-expressing the B cell receptors and CARscomprises culturing T cells transduced or transfected with nucleic acidsencoding B cell receptors and CARs in culture media. Media for culturingT cells are well known to those of skill in the art. In someembodiments, T cells are cultured in DMEM or RPMI medium. In someembodiments, the medium is supplemented with FBS, e.g, 5-20% FBS, e.g.,10% FBS. In some embodiments, the medium is supplemented with HEPES,e.g., 1-100 mM HEPES, e.g., 10 mM HEPES. In some embodiments, the mediumis supplemented with penicillin, e.g., 10-500 U/ml penicillin, e.g., 100U/ml penicillin. In some embodiments, the medium is supplemented withstreptomycin, e.g., 10-500 ug/ml streptomycin, e.g., 100 ug/mlstreptomycin. In some embodiments, the medium is supplemented withL-alanyl-L-glutamine, e.g., 0.1-10 mM L-alanyl-L-glutamine, e.g., 2 mML-alanyl-L-glutamine.

In some embodiments, measuring the level of T cell activation comprisesmeasuring the nucleic acid or protein level of a gene expressed inactivated T cells. Examples of genes downregulated during T cellactivation include, for example, L-selectin, CD127, and BCL-2. Examplesof genes downregulated during T cell activation include, for exampleCD69, CD25, CD40L, CD44, Ki67, and KLRG1. In some embodiments, the Tcell comprises a fluorescent protein reporter gene under the control ofa transcription factor that activates transcription when the T cell isactivated and measuring activation comprises measuring the amount offluorescent protein produced. In some embodiments, the transcriptionfactor is Jun, NF-κB or Rel.

Gene expression can be measured at either the RNA or protein level.Assays for detecting RNA include, but are not limited to, Northern blotanalysis, RT-PCR, sequencing technology, RNA in situ hybridization(using e.g., DNA or RNA probes to hybridize RNA molecules present in thesample), in situ RT-PCR (e.g., as described in Nuovo G J, et al. Am JSurg Pathol. 1993, 17: 683-90; Komminoth P, et al. Pathol Res Pract.1994, 190: 1017-25), and oligonucleotide microarray (e.g., byhybridization of polynucleotide sequences derived from a sample tooligonucleotides attached to a solid surface (e.g., a glass wafer withaddressable location, such as Affymetrix microarray (Affymetrix®, SantaClara, Calif.)).

Assays for detecting protein levels include, but are not limited to,immunoassays (also referred to herein as immune-based or immuno-basedassays, e.g., Western blot, ELISA, proximity extension assays, andELISpot assays), Mass spectrometry, and multiplex bead-based assays.Other examples of protein detection and quantitation methods includemultiplexed immunoassays as described for example in U.S. Pat. Nos.6,939,720 and 8,148,171, and published U.S. Patent Application No.2008/0255766, and protein microarrays as described for example inpublished U.S. Patent Application No. 2009/0088329.

In some embodiments, once an activated T cell is identified, the CARexpressed in the T cell is identified. Accordingly, in some embodiments,protocols for identifying activated T cells allow for the identificationof activated T cells and the separation of activated T cells fromunactivated T cells. One example of such a protocol is flow cytometry.The use of flow cytometry generally, and Fluorescence-activated cellsorting (FACS) in particular, are readily known to those of skill in theart for the purpose of cell sorting based on a variety of properties. InFACS, a heterogeneous mixture of biological cells can be sorted into twoor more containers, one cell at a time, based upon the specific lightscattering and fluorescent characteristics of each cell. This allows,for example, for cells to be sorted on the basis of fluorescent markers.Accordingly, in certain embodiments, T cell activation can be measuredby levels of a fluorescently marked or labeled transcript or protein. Insome embodiments, the expression level of a protein, e.g., a cellsurface localized protein, e.g., a protein upregulated or downregulatedin activated T cells described herein, can be measured by contacting thecells with an antibody coupled, covalently or non-covalently, to afluorescent label. In some embodiments, the antibody targets the proteinupregulated or downregulated in activated T cells. This can allow thecells to be sorted based on expression level of a protein upregulated ordownregulated in activated T cells, thereby allowing separation ofactivated from unactivated T cells. In one exemplary embodiment,activated T cells can be identified by binding of the T cells toGFP-labeled anti-CD69 antibody.

In some embodiments, detecting binding between a putative B cellreceptor ligand and a cell expressing a B cell receptor comprisesvisualizing binding of the putative B cell receptor ligand to the cellexpressing the B cell receptor. For example, in some embodiments, theligand is tagged to allow for visualization of the localization of theligand. Suitable tags include, for example, fluorescent genes such asGFP, YFP, RFP and the like. In some embodiments, localization of theputative B cell receptor ligand to the cell expressing the B cellreceptor can be assessed using any suitable method known by those ofskill in the art, e.g., fluorescence microscopy, immunohistochemistry,or FACS. In some embodiments, a B cell receptor ligand binds to thecells expressing the B cell receptor and does not bind to the same celltype when the B cell receptor is not expressed.

In some embodiments the library pf putative B cell receptor ligands iscontacted to the B cell receptor by phage display. “Phage display” is atechnique by which variant polypeptides are displayed as fusion proteinsto a coat protein on the surface of phage, e.g. filamentous phage,particles. A utility of phage display lies in the fact that largelibraries of randomized protein variants can be rapidly and efficientlysorted for those sequences that bind to a target molecule with highaffinity. Display of peptides and proteins libraries on phage has beenused for screening millions of polypeptides for ones with specificbinding properties. Polyvalent phage display methods have been used fordisplaying small random peptides and small proteins through fusions toeither gene 111 or gene VIII of filamentous phage. Wells and Lowman,Curr. Opin. Struct. Biol., 1992, 3:355-362 and references cited therein.In monovalent phage display, a protein or peptide library is fused to agene 111 or a portion thereof and expressed at low levels in thepresence of wild type gene III protein so that phage particles displayone copy or none of the fusion proteins. Avidity effects are reducedrelative to polyvalent phage so that sorting is on the basis ofintrinsic ligand affinity, and phagemid vectors are used, which simplifyDNA manipulations. Lowman and Wells, Methods: A companion to Methods inEnzymology, 1991, 3:205-216.

Phage display of proteins, peptides and mutated variants thereof,including constructing a family of variant replicable vectors containinga transcription regulatory element operably linked to a gene fusionencoding a fusion polypeptide, transforming suitable host cells,culturing the transformed cells to form phage particles which displaythe fusion polypeptide on the surface of the phage particle, contactingthe recombinant phage particles with a target molecule so that at leasta portion of the particle bind to the target, separating the particleswhich bind from those that do not are known and may be used with thetransformation method of the invention. See U.S. Pat. No. 5,750,373; WO97/09446; U.S. Pat. Nos. 5,514,548; 5,498,538; 5,516,637; 5,432,018; WO96/22393; U.S. Pat. Nos. 5,658,727; 5,627,024; WO 97/29185; O'Boyle etal, 1997, Virology, 236:338-347; Soumillion et al, 1994, Appl. Biochem.Biotech., 47:175-190; O'Neil and Hoess, 1995, Curr. Opin. Struct. Biol.,5:443-449; Makowski, 1993, Gene, 128:5-11; Dunn, 1996, Curr. Opin.Struct. Biol., 7:547-553; Choo and King, 1995, Curr. Opin. Struct.Biol., 6:431-436; Bradbury and Cattaneo, 1995, TINS, 18:242-249; Corteseet al., 1995, Curr. Opin. Struct. Biol., 6:73-80; Allen et al., 1995,TIBS, 20:509-516; Lindquist and Naderi, 1995, FEMS Micro. Rev.,17:33-39; Clarkson and Wells, 1994, Tibtech, 12:173-184; Barbas, 1993,Curr. Opin. Biol., 4:526-530; McGregor, 1996, Mol. Biotech., 6:155-162;Cortese et al., 1996, Curr. Opin. Biol., 7; 616-621; McLafferty et al.,1993, Gene, 128:29-36. Using phage display, in some embodiments,putative B cell receptor ligands capable of binding to the B cellreceptor as described herein are isolated from a suitable library.Exemplary putative B cell receptor ligand libraries includephage-peptide libraries such as New England Biolabs Ph.D.-7 and Ph.D.-12libraries. Methods of generating peptide libraries and screening theselibraries are also disclosed in U.S. Pat. Nos. 5,723,286; 5,432,018;5,580,717; 5,427,908; and 5,498,530. See also U.S. Pat. Nos. 5,770,434;5,734,018; 5,698,426; 5,763,192; and 5,723,323. In the selectionprocess, a putative B cell receptor ligand library can be probed withthe target B cell receptor or a fragment thereof and members of thelibrary that are capable of binding to the B cell receptor can beisolated, typically by retention on a support. Such screening processmay be performed by multiple rounds (e.g., including both positive andnegative selections) to enrich the pool of putative B cell receptorligands capable of binding to the B cell receptor. In some embodiments,negative selection is performed in each round of panning. Individualclones of the enriched pool can then be isolated and furthercharacterized to identify those having desired binding activity andbiological activity. Sequences of the putative B cell receptor ligandscan also be determined via conventional methodology.

As an example, phage displays typically use a covalent linkage to bindthe protein (e.g., putative B cell receptor ligand domain) component toa bacteriophage coat protein. The linkage results from translation of anucleic acid encoding the putative B cell receptor ligand domaincomponent fused to the coat protein. The linkage can include a flexiblepeptide linker, a protease site, or an amino acid incorporated as aresult of suppression of a stop codon. Phage display is described, forexample, in U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317;WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999) J. Biol. Chem274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20;Hoogenboom et al. (2000) Immunol Today 2:371-8 and Hoet et al. (2005)Nat Biotechnol. 23(3)344-8. Bacteriophage displaying the putative B cellreceptor ligand domain component can be grown and harvested usingstandard phage preparatory methods, e.g. PEG precipitation from growthmedia. After selection of individual display phages, the nucleic acidencoding the selected protein components can be isolated from cellsinfected with the selected phages or from the phage themselves, afteramplification. Individual colonies or plaques can be selected, and thenthe nucleic acid may be isolated and sequenced.

After display library members are isolated for binding to the targetantigen, each isolated library member can be also tested for its abilityto bind to a non-target molecule to evaluate its binding specificity.Examples of non-target molecules include streptavidin on magnetic beads,blocking agents such as bovine serum albumin, non-fat bovine milk, soyprotein, any capturing or target immobilizing monoclonal antibody, ornon-transfected cells which do not express the target. A high-throughputELISA screen can be used to obtain the data, for example. The ELISAscreen can also be used to obtain quantitative data for binding of eachlibrary member to the target as well as for cross species reactivity torelated targets or subunits of the target antigen and also underdifferent condition such as pH 6 or pH 7.5. The non-target and targetbinding data are compared (e.g., using a computer and software) toidentify library members that specifically bind to the target.

Putative B Cell Receptor Ligands

Provided herein are methods of identifying unique B cell receptorligands, e.g., for cancer therapy, comprising identifying a putativeunique B cell receptor ligand as binding a unique B cell receptor.

In some embodiments, the putative B cell receptor ligand comprises apolypeptide. In some embodiments, a putative B cell receptor ligandcomprises a cyclopeptide. In some embodiments, a putative B cellreceptor ligand comprises a peptoid. In some embodiments, a putative Bcell receptor ligand comprises a cyclopeptoid. In some embodiments, theputative B cell receptor ligand comprises a polysaccharide. In someembodiments, the putative B cell receptor ligand comprises a lipid. Insome embodiments, the putative B cell receptor ligand comprises a smallmolecule.

In some embodiments, the putative B cell receptor ligand comprises anamino acid sequence that encodes a portion or all of a cellular protein.In some embodiments, the putative B cell receptor ligand comprises anamino acid sequence that does not encode a portion or all of a cellularprotein.

In some embodiments, the putative B cell receptor ligand is 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40amino acids in length, e.g., 9 amino acids in length. In someembodiments, the putative B cell receptor ligand is less than 20, lessthan 15, or less than 10 amino acids in length. In some embodiments, theputative B cell receptor ligand is 2-20, 5-15, or 7-10 amino acids inlength.

In some embodiments, the putative B cell receptor ligand comprises thesequence YX_(n)Z. In some embodiments, Y and Z are polar uncharged aminoacids. In some embodiments, Y and Z are C or conservative substitutionsof C, e.g., S, A, M, or T. In some embodiments, the putative B cellreceptor ligand comprises the sequence CX_(n)C. In some embodiments, theputative B cell receptor ligand comprises the sequence SX_(n)S. In someembodiments, the putative B cell receptor ligand comprises the sequenceCX_(n)S. In some embodiments, the putative B cell receptor ligandcomprises the sequence SX_(n)C. In some embodiments, X is any of the 20amino acids encoded by DNA. In some embodiments, n is 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., n is 7. Insome embodiments, n is 15 or less, 12 or less, or 9 or less. In someembodiments, n is 2-15, 5-10, or 6-8. In some embodiments, the putativeB cell receptor ligand comprises any of SEQ ID NOs: 1-3.

In some embodiments, the putative B cell receptor ligand comprises acyclopeptide with the sequence CX_(n)C, and the N- and C-terminal Cysform a Cys-Cys interaction, circularizing the cyclopeptide.

Also provided herein are libraries of putative B cell receptor ligands.

In some embodiments, the library of putative B cell receptor ligands isgenerated from a cDNA library and with each putative B cell receptorligand comprising a portion or all of a cDNA.

In some embodiments, the library of putative B cell receptor ligandscomprises a peptide library. In some embodiments, the peptide library isa combinatorial peptide library. In some embodiments, the putative Bcell receptor ligands in the peptide library comprises the sequenceYX_(n)Z with the putative B cell receptor ligands differing in X_(n)sequence. In some embodiments, Y and Z are polar uncharged amino acids.In some embodiments, Y and Z are C or conservative substitutions of C,e.g., S, A, M, or T. In some embodiments, the putative B cell receptorligands in the peptide library comprises the sequence CX_(n)C with theputative B cell receptor ligands differing in X_(n) sequence. In someembodiments, the putative B cell receptor ligands in the peptide librarycomprises the sequence SX_(n)S with the putative B cell receptor ligandsdiffering in X_(n) sequence. In some embodiments, the putative B cellreceptor ligands in the peptide library comprises the sequence CX_(n)Swith the putative B cell receptor ligands differing in X_(n) sequence.In some embodiments, the putative B cell receptor ligands in the peptidelibrary comprises the sequence SX_(n)C with the putative B cell receptorligands differing in X_(n) sequence. In some embodiments, X is any ofthe 20 amino acids encoded by DNA. In some embodiments, n is 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., n is7. In some embodiments, n is 15 or less, 12 or less, or 9 or less. Insome embodiments, n is 2-15, 5-10, or 6-8. In some embodiments, X_(n)sequence is generated by PCR with oligonucleotides having degenerateNNN, NNK, or NNS codons at the X positions. In some embodiments, thedegenerate codons are NNK codons.

In some embodiments the putative B cell receptor ligand comprises theantigen binding domain of a CAR. In some embodiments the putative B cellreceptor ligand is linked to a phage, e.g., as a component of a phagedisplay libaray.

Chimeric Antigen Receptors (CARs)

Disclosed herein are methods for identifying B cell receptor ligands byco-expressing B cell receptors and CARs having a putative B cellreceptor ligand domain as an extracellular domain and measuring T cellactivation.

Also disclosed herein are methods for treating cancer by treating asubject with CAR-expressing T-cells, wherein the CAR comprises anantigen binding domain that specifically binds a cancer-specific antigenin a cancer-specific manner and a vaccine comprising a polypeptide or anucleic acid expressing the cancer-specific antigen, or acancer-specific fragment thereof.

In one aspect an exemplary CAR construct disclosed herein comprise anoptional leader sequence, an extracellular putative B cell receptorligand domain, a hinge, a transmembrane domain, and an intracellularstimulatory domain. In one aspect an exemplary CAR construct comprisesan optional leader sequence, an extracellular putative B cell receptorligand domain, a hinge, a transmembrane domain, an intracellularcostimulatory domain and an intracellular stimulatory domain.

The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers toa recombinant polypeptide construct comprising at least an extracellularligand domain, a transmembrane domain and a cytoplasmic signaling domain(also referred to herein as “an intracellular signaling domain”)comprising a functional signaling domain derived from a stimulatorymolecule as defined below. In some embodiments, the domains in the CARpolypeptide construct are in the same polypeptide chain, e.g., comprisea chimeric fusion protein. In some embodiments, the domains in the CARpolypeptide construct are not contiguous with each other.

Antigen Binding Domain

In some embodiments, the CAR described herein comprises an extracellulardomain. In some embodiments, the extracellular domain comprises anantigen binding domain. In some embodiments, the antigen binding domainis a putative B cell receptor ligand domain comprising a putative B cellreceptor ligand, e.g., a putative B cell receptor ligand describedherein. In some embodiments, provided herein are a library of CARs withthe CARs differing in their antigen binding domains, e.g., putative Bcell receptor ligand domains. In some embodiments, each CAR within thelibrary comprises a distinct antigen binding domain, e.g., putative Bcell receptor ligand domain. In some embodiments, the library of CARscomprises an extracellular domain and the extracellular domain comprisesthe library of antigen binding domains, e.g., putative B cell receptorligands described herein.

In some embodiments, the putative B cell receptor ligand domain furthercomprises an Fc domain, which is CH2 and CH3 of a heavy chain constantregion. In some embodiments, the Fc domain is from a heavy chainconstant region chosen from, e.g., the heavy chain constant regions ofIgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly,chosen from, e.g., the (e.g., human) heavy chain constant regions ofIgG1, IgG2, IgG3, and IgG4.

In some embodiments, antigen binding domain comprises an immunoglobulinchain or fragment thereof, comprising at least one immunoglobulinvariable domain sequence. The term “antigen binding domain” encompassesantibodies and antibody fragments. In an embodiment, an antibodymolecule is a multispecific antibody molecule, e.g., it comprises aplurality of immunoglobulin variable domain sequences, wherein a firstimmunoglobulin variable domain sequence of the plurality has bindingspecificity for a first epitope and a second immunoglobulin variabledomain sequence of the plurality has binding specificity for a secondepitope. In an embodiment, a multispecific antibody molecule is abispecific antibody molecule. A bispecific antibody has specificity forno more than two antigens. A bispecific antibody molecule ischaracterized by a first immunoglobulin variable domain sequence whichhas binding specificity for a first epitope and a second immunoglobulinvariable domain sequence that has binding specificity for a secondepitope.

In some embodiments, the antigen binding domain specifically binds acancer-specific antigen.

In some embodiments, the CARs of the present invention includes CARscomprising an antigen binding domain (e.g., antibody or antibodyfragment) that binds to a MHC presented peptide. Normally, peptidesderived from endogenous proteins fill the pockets of Majorhistocompatibility complex (MHC) class I molecules, and are recognizedby T cell receptors (TCRs) on CD8+T lymphocytes. The MHC class Icomplexes are constitutively expressed by all nucleated cells. Incancer, virus-specific and/or tumor-specific peptide/MHC complexesrepresent a unique class of cell surface targets for immunotherapy.TCR-like antibodies targeting peptides derived from viral or tumorantigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2have been described (see, e.g., Sastry et al., J Virol. 201185(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Vermaet al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33;Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example,TCR-like antibody can be identified from screening a library, such as ahuman scFv phage displayed library.

The antigen binding domain can be any protein that binds to the antigenincluding but not limited to a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody, and a functional fragment thereof, including but not limitedto a single-domain antibody such as a heavy chain variable domain (VH),a light chain variable domain (VL) and a variable domain (VHH) ofcamelid derived nanobody, and to an alternative scaffold known in theart to function as antigen binding domain, such as a recombinantfibronectin domain, and the like. In some instances, it is beneficialfor the antigen binding domain to be derived from the same species inwhich the CAR will ultimately be used in. For example, for use inhumans, it may be beneficial for the antigen binding domain of the CARto comprise human or humanized residues for the antigen binding domainof an antibody or antibody fragment.

In one aspect, the antigen binding domain comprises a human antibody oran antibody fragment.

In one aspect, the antigen binding domain comprises a humanized antibodyor an antibody fragment.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, InternationalPublication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002),Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods,20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84(1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto etal., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., CancerRes., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), andPedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which isincorporated herein in its entirety by reference. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, for exampleimprove, antigen binding. These framework substitutions are identifiedby methods well-known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature,332:323, which are incorporated herein by reference in theirentireties.)

A humanized antibody or antibody fragment has one or more amino acidresidues remaining in it from a source which is nonhuman. These nonhumanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. As providedherein, humanized antibodies or antibody fragments comprise one or moreCDRs from nonhuman immunoglobulin molecules and framework regionswherein the amino acid residues comprising the framework are derivedcompletely or mostly from human germline. Multiple techniques forhumanization of antibodies or antibody fragments are well-known in theart and can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized antibodies and antibody fragments, substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a nonhuman species. Humanized antibodies areoften human antibodies in which some CDR residues and possibly someframework (FR) residues are substituted by residues from analogous sitesin rodent antibodies. Humanization of antibodies and antibody fragmentscan also be achieved by veneering or resurfacing (EP 592,106; EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al.,PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332),the contents of which are incorporated herein by reference herein intheir entirety.

In some embodiments, an antigen binding domain is derived from a displaylibrary. A display library is a collection of entities; each entityincludes an accessible polypeptide component and a recoverable componentthat encodes or identifies the polypeptide component. The polypeptidecomponent is varied so that different amino acid sequences arerepresented. The polypeptide component can be of any length, e.g. fromthree amino acids to over 300 amino acids. A display library entity caninclude more than one polypeptide component, for example, the twopolypeptide chains of a Fab. In one exemplary embodiment, a displaylibrary can be used to identify an antigen binding domain. In aselection, the polypeptide component of each member of the library isprobed with the antigen, or a fragment there, and if the polypeptidecomponent binds to the antigen, the display library member isidentified, typically by retention on a support.

Retained display library members are recovered from the support andanalyzed. The analysis can include amplification and a subsequentselection under similar or dissimilar conditions. For example, positiveand negative selections can be alternated. The analysis can also includedetermining the amino acid sequence of the polypeptide component andpurification of the polypeptide component for detailed characterization.

A variety of formats can be used for display libraries. Examples includethe phage display. In phage display, the protein component is typicallycovalently linked to a bacteriophage coat protein. The linkage resultsfrom translation of a nucleic acid encoding the protein component fusedto the coat protein. The linkage can include a flexible peptide linker,a protease site, or an amino acid incorporated as a result ofsuppression of a stop codon. Phage display is described, for example, inU.S. Pat. No. 5,223,409; WO 92/18619; WO 91/17271; WO 92/20791; WO92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809.Bacteriophage displaying the protein component can be grown andharvested using standard phage preparatory methods, e.g. PEGprecipitation from growth media. After selection of individual displayphages, the nucleic acid encoding the selected protein components can beisolated from cells infected with the selected phages or from the phagethemselves, after amplification. Individual colonies or plaques can bepicked, the nucleic acid isolated and sequenced.

Other display formats include cell based display (see, e.g., WO03/029456), protein-nucleic acid fusions (see, e.g., U.S. Pat. No.6,207,446), ribosome display, and E. coli periplasmic display.

In one aspect the CAR comprises a leader sequence at the amino-terminus(N-ter) of the antigen binding domain. In one aspect, the CAR furthercomprises a leader sequence at the N-terminus of the antigen bindingdomain, wherein the leader sequence is optionally cleaved from theantigen binding domain (e.g., aa scFv) during cellular processing andlocalization of the CAR to the cellular membrane. In some embodiments,the leader sequence is an interleukin 2 signal peptide.

Transmembrane Domain

The transmembrane domain may be derived either from a natural or from arecombinant source. Where the source is natural, the domain may bederived from any membrane-bound or transmembrane protein. In one aspectthe transmembrane domain is capable of signaling to the intracellulardomain(s) whenever the CAR has bound to a target. A transmembrane domainof particular use in this invention may include at least thetransmembrane region(s) of e.g., the alpha, beta or zeta chain of theT-cell receptor, CD28, CD3 epsilon, CD45, CD4, CDS, CD8 (e.g., CD8alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134,CD137, CD154. In some embodiments, a transmembrane domain may include atleast the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27,LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR,HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19,IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D,ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, rfGAL, CD11a, LFA-1,ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7,TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D),SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C, and CD19.

In some instances, the transmembrane domain can be attached to theextracellular region of the CAR, e.g., the ligand domain of the CAR, viaa hinge, e.g., a hinge from a human protein. For example, in oneembodiment, the hinge can be a human Ig (immunoglobulin) hinge, e.g., anIgG4 hinge, or a CD8a hinge.

Cytoplasmic Domain

The cytoplasmic domain or region of the present CAR includes anintracellular signaling domain. An intracellular signaling domain iscapable of activation of at least one of the normal effector functionsof the immune cell in which the CAR has been introduced.

Examples of intracellular signaling domains for use in the CAR of theinvention include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any recombinant sequence that has thesame functional capability.

T cell activation can be said to be mediated by two distinct classes ofcytoplasmic signaling sequences: those that initiate antigen-dependentprimary activation through the TCR (primary intracellular signalingdomains) and those that act in an antigen-independent manner to providea secondary or costimulatory signal (secondary cytoplasmic domain, e.g.,a costimulatory domain).

An “intracellular signaling domain,” as the term is used herein, refersto an intracellular portion of a molecule. The intracellular signalingdomain can generate a signal that promotes an immune effector functionof the CAR containing cell, e.g., a CART cell or CAR-expressing NK cell.Examples of immune effector function, e.g., in a CART cell orCAR-expressing NK cell, include cytolytic activity and helper activity,including the secretion of cytokines. In embodiments, the intracellularsignal domain transduces the effector function signal and directs thecell to perform a specialized function. While the entire intracellularsignaling domain can be employed, in many cases it is not necessary touse the entire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the intracellular signaling domainsufficient to transduce the effector function signal. In an embodiment,the intracellular signaling domain can comprise a primary intracellularsignaling domain. Exemplary primary intracellular signaling domainsinclude those derived from the molecules responsible for primarystimulation, or antigen dependent simulation. In an embodiment, theintracellular signaling domain can comprise a costimulatoryintracellular domain. Exemplary costimulatory intracellular signalingdomains include those derived from molecules responsible forcostimulatory signals, or antigen independent stimulation. For example,in the case of a CAR-expressing immune effector cell, e.g., CART cell orCAR-expressing NK cell, a primary intracellular signaling domain cancomprise a cytoplasmic sequence of a T cell receptor, and acostimulatory intracellular signaling domain can comprise cytoplasmicsequence from co-receptor or costimulatory molecule.

A primary intracellular signaling domain can comprise a signaling motifwhich is known as an immunoreceptor tyrosine-based activation motif orITAM. Examples of FFAM containing primary cytoplasmic signalingsequences include, but are not limited to, those derived from CD3 zeta,FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, CD278 (“ICOS”), FceRI, CD66d, DAP10, and DAP12.

The intracellular signalling domain of the CAR can comprise the primarysignalling domain, e.g., CD3-zeta signaling domain, by itself or it canbe combined with any other desired intracellular signaling domain(s)useful in the context of a CAR of the invention. For example, theintracellular signaling domain of the CAR can comprise a primarysignalling domain, e.g., CD3 zeta chain portion, and a costimulatorysignaling domain.

A costimulatory intracellular signaling domain refers to theintracellular portion of a costimulatory molecule. The intracellularsignaling domain can comprise the entire intracellular portion, or theentire native intracellular signaling domain, of the molecule from whichit is derived, or a functional fragment thereof. The term “costimulatorymolecule” refers to the cognate binding partner on a T cell thatspecifically binds with a costimulatory ligand, thereby mediating acostimulatory response by the T cell, such as, but not limited to,proliferation. Costimulatory molecules are cell surface molecules otherthan antigen receptors or their ligands that are required for anefficient immune response. Examples of such molecules include a MHCclass I molecule, TNF receptor proteins, Immunoglobulin-like proteins,cytokine receptors, integrins, signaling lymphocytic activationmolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1,LFA-1 (CD1 la/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278),GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44,NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, CD11c,ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligandthat specifically binds with CD83. For example, CD27 co-stimulation hasbeen demonstrated to enhance expansion, effector function, and survivalof human CART cells in vitro and augments human T cell persistence andantitumor activity in vivo (Song et al. Blood. 2012; 119(3):696-706).

Expression in Cells

In some embodiments, the methods described herein comprise expressing Bcell receptors and putative B cell receptor ligands, e.g., CARscomprising putative B cell receptor ligands, in cells, e.g., T cells foridentifying a B cell receptor ligand, e.g., for treatment of cancer. Themethods described herein also comprise expressing CARs in T cells forcancer treatment.

In some embodiments, the disclosure encompasses DNA constructs forexpressing CARs in cells, e.g., T cells. The nucleic acid sequencescoding for the desired molecules can be obtained using recombinantmethods known in the art, such as, for example by screening librariesfrom cells expressing the gene, by deriving the gene from a vector knownto include the same, or by isolating directly from cells and tissuescontaining the same, using standard techniques. For example, as isdescribed herein, sequences of B cell receptors can be derived fromcancer cells. Recombinant DNA and molecular cloning techniques used hereare well known in the art and are described, for example, by Sambrook,J., Fritsch, E. F. and Maniatis, T. MOLECULAR CLONING: A LABORATORYMANUAL, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor,N.Y., 1989 (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M.L. and Enquist, L. W. EXPERIMENTS WITH GENE FUSIONS; Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. etal., IN CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, published by GreenePublishing and Wiley-Interscience, 1987; (the entirety of each of whichis hereby incorporated herein by reference).

Alternatively, the gene of interest can be produced synthetically,rather than cloned.

The present disclosure also provides vectors in which a DNA of thepresent disclosure is inserted. Vectors derived from retroviruses suchas the lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity. In another embodiment, the desired B cell receptor orCAR can be expressed in the cells by way of transposons.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lenti viruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lenti viruses. Vectors derived from lenti virusesoffer the means to achieve significant levels of gene transfer in vivo.

Expression of natural or synthetic nucleic acids encoding B cellreceptors and CARs is typically achieved by operably linking a nucleicacid encoding the polypeptide expressing the B cell receptor or CAR orportions thereof to a promoter, and incorporating the construct into anexpression vector. The vectors can be suitable for replication andintegration into eukaryotes. Typical cloning vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the desired nucleicacid sequence. The expression constructs of the disclosure may also beused for nucleic acid immunization and gene therapy, using standard genedelivery protocols. Methods for gene delivery are known in the art. See,e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, thedisclosure provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, retrovirus vectorsare used. A number of retrovirus vectors are known in the art. In someembodiments, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Factor-1a (EF-1a).However, other constitutive promoter sequences may also be used,including, but not limited to the simian virus 40 (SV40) early promoter,mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV)long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemiavirus promoter, an Epstein-Barr virus immediate early promoter, a Roussarcoma virus promoter, as well as human gene promoters such as, but notlimited to, the actin promoter, the myosin promoter, the hemoglobinpromoter, and the creatine kinase promoter. Further, the disclosure isnot limited to the use of constitutive promoters. Inducible promotersare also contemplated as part of the disclosure. The use of an induciblepromoter provides a molecular switch capable of turning on expression ofthe polynucleotide sequence which it is operatively linked when suchexpression is desired, or turning off the expression when expression isnot desired. Examples of inducible promoters include, but are notlimited to a metallothionine promoter, a glucocorticoid promoter, aprogesterone promoter, and a tetracycline promoter. In some embodiments,the promoter is a EF-1a promoter.

In order to assess the expression of a B cell receptors or CAR orportions thereof, the expression vector to be introduced into a cell canalso contain either a selectable marker gene or a reporter gene or bothto facilitate identification and selection of expressing cells. In otheraspects, the selectable marker may be carried on a separate piece of DNAand used in a co-transfection procedure. Both selectable markers andreporter genes may be flanked with appropriate regulatory sequences toenable expression in the host cells. Useful selectable markers include,for example, antibiotic-resistance genes, such as neo and the like, andfluorescent genes such as GFP, YFP, RFP and the like. In someembodiments, reporter genes or selectable marker genes are excluded froma CAR polypeptide used in a therapy as described herein.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity, antibiotic resistance or fluorescence. Expression ofthe reporter gene is assayed at a suitable time after the DNA has beenintroduced into the recipient cells. Suitable reporter genes may includegenes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescentprotein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).Suitable expression systems are well known and may be prepared usingknown techniques or obtained commercially. In general, the constructwith the minimal 5′ flanking region showing the highest level ofexpression of reporter gene is identified as the promoter. Such promoterregions may be linked to a reporter gene and used to evaluate agents forthe ability to modulate promoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans. In some embodiments, the host cell is a T cell.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexample of a colloidal system for use as a delivery vehicle in vitro andin vivo is a liposome (e.g., an artificial membrane vesicle).

Sources of Cells

In some embodiments, cells are transfected with nucleic acids expressinga B cell receptor and/or a CAR. The term “transfected” or “transformed”or “transduced” as used herein refers to a process by which exogenousnucleic acid is transferred or introduced into the host cell. A“transfected” or “transformed” or “transduced” cell is one which hasbeen transfected, transformed or transduced with exogenous nucleic acid.The cell includes the primary subject cell and its progeny.

In some embodiments, the cells are mammalian cells. In some embodiments,the cells are human cells. In some embodiments, the cells are immunecells, e.g., B cells, T cells, or NK cells. In particular embodiments,the cells are T cells.

Immune cells (e.g., T cells) can be obtained from a number of sources,including peripheral blood mononuclear cells, bone marrow, lymph nodetissue, cord blood, thymus tissue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. The immune cells(e.g., T cells) may also be generated from induced pluripotent stemcells or hematopoietic stem cells or progenitor cells. In someembodiments, any number of immune cell lines, including but not limitedto T cell lines, including, for example, Hep-2, Jurkat, and Raji celllines, available in the art, may be used. In some embodiments, immunecells (e.g., T cells) can be obtained from a unit of blood collectedfrom a subject using any number of techniques known to the skilledartisan, such as Ficoll™ separation. In some embodiments, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, NK cells, other nucleated white bloodcells, red blood cells, and platelets. In some embodiments, the cellscollected by apheresis may be washed to remove the plasma fraction andto place the cells in an appropriate buffer or media for subsequentprocessing steps. In some embodiments, the cells are washed withphosphate buffered saline (PBS). In an alternative embodiment, the washsolution lacks calcium and may lack magnesium or may lack many if notall divalent cations. Again, surprisingly, initial activation steps inthe absence of calcium lead to magnified activation. As those ofordinary skill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In some embodiments, immune cells (e.g., T cells) are isolated fromperipheral blood lymphocytes by lysing the red blood cells and depletingthe monocytes, for example, by centrifugation through a PERCOLL™gradient or by counterflow centrifugal elutriation. A specificsubpopulation of T cells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, andCD45RO⁺T cells, can be further isolated by positive or negativeselection techniques.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺.

Alternatively, in certain embodiments, T regulatory cells are depletedby anti-C25 conjugated beads or other similar method of selection.

Methods of Treatment

Provided herein are methods of treatment using the B cell receptorligands identified herein. In particular, provided herein are methodsfor rapid treatment of B cell malignancies. For example, the methodsdescribed herein allow for the rapid identification of a B cell receptorligand by co-expressing a CAR having a putative B cell receptor ligandand a B cell receptor in a T cell, and identifying binding of theputative B cell receptor ligand to the B cell receptor by activation ofthe B cell, and in some embodiments, the same CAR used in identificationof the B cell receptor ligand can be used for treatment, allowing forthe rapid identification and treatment of B cell malignancies. In someembodiments, provided herein are methods of treatment using B cellreceptor ligands that activate a T cell when a CAR comprising the B cellligand is co-expressed with the B cell receptor of the lymphoma cells ofa subject being treated in T cells.

In some embodiments, a subject is treated with a B cell receptor ligandcoupled to a therapeutic agent.

In some embodiments, the B cell receptor ligand coupled to a therapeuticagent comprises a therapeutic CAR, e.g., a CAR described herein,expressed in a T cell as is described herein, e.g., a CAR-T cell. Insome embodiments, the therapeutic CAR comprises a CAR used in a methodof identifying a B cell receptor.

In some embodiments, the CART cell, e.g., a T cell expressing a CARdescribed herein, results in greater specificity and/or activity than acontrol. In some embodiments, the control comprises a CAR T cell. Insome embodiments, the CAR T cell has an antigen binding domain specificfor an antigen unrelated to cancer. In some embodiments, the CAR T cellhas an antigen binding domain specific for a cancer-specific antigen, asis described herein.

In some embodiments, activity and specificity can be demonstrated bycytotoxicity. In some embodiments, activity comprises cytotoxicity,e.g., as measured by % lysis, towards cells expressing the unique B cellreceptor relative to a control. In some embodiments, the % lysis is0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or more greaterthan a control.

In some embodiments, specificity comprises cytotoxicity, e.g., asmeasured by % lysis, towards cells that do not express the unique B cellreceptor. In some embodiments, the % lysis is 0.1%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, or more less than a control. In someembodiments, % lysis is measured at an effector:target ratio of 1:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or greater.

In some embodiments, subjects treated with the CART cell, e.g., a T cellexpressing a CAR described herein, exhibit reduced cytokine releasesyndrome (CRS) relative to a subject treated with a control.

As used herein, “coupled” refers to the association of two moleculesthough covalently and non-covalent interactions, e.g., by hydrogen,ionic, or Van-der-Waals bonds. Such bonds may be formed between at leasttwo of the same or different atoms or ions as a result of redistributionof electron densities of those atoms or ions. For example, a B cellligand may be coupled to a therapeutic agent as a fusion protein.

In some embodiments, a therapeutic agent comprises a radioactive isotopesuch as an α-, β-, or γ-emitter, or a β- and γ-emitter.

In some embodiments, a therapeutic agent comprises a chemotherapy.Chemotherapeutic agents include, for example, including alkylatingagents, anthracyclines, cytoskeletal disruptors (Taxanes), epothilones,histone deacetylase inhibitors, inhibitors of topoisomerase I,inhibitors of topoisomerase II, kinase inhibitors, nucleotide analogsand precursor analogs, peptide antibiotics, platinum-based agents,retinoids, vinca alkaloids and derivatives thereof. Non-limitingexamples include: (i) anti-angiogenic agents (e.g., TNP-470, plateletfactor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TIMP1and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment ofplasminogen), endostatin, bFGF soluble receptor, transforming growthfactor beta, interferon alpha, soluble KDR and FLT-1 receptors,placental proliferin-related protein, as well as those listed byCarmeliet and Jain (2000)); (ii) a VEGF antagonist or a VEGF receptorantagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGFreceptor fragments, aptamers capable of blocking VEGF or VEGFR,neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinasesand any combinations thereof; and (iii) chemotherapeutic compounds suchas, e.g., pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine,gemcitabine and cytarabine), purine analogs, folate antagonists andrelated inhibitors (mercaptopurine, thioguanine, pentostatin and2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitoticagents including natural products such as vinca alkaloids (vinblastine,vincristine, and vinorelbine), microtubule disruptors such as taxane(paclitaxel, docetaxel), vincristine, vinblastine, nocodazole,epothilones, and navelbine, epidipodophyllotoxins (etoposide andteniposide), DNA damaging agents (actinomycin, amsacrine,anthracyclines, bleomycin, busulfan, camptothecin, carboplatin,chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin,daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin,iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone,nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide,triethylenethiophosphoramide and etoposide (VP16)); antibiotics such asdactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin),idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin(mithramycin) and mitomycin; enzymes (L-asparaginase which systemicallymetabolizes L-asparagine and deprives cells which do not have thecapacity to synthesize their own asparagine); antiplatelet agents;antiproliferative/antimitotic alkylating agents such as nitrogenmustards (mechlorethamine, cyclophosphamide and analogs, melphalan,chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine andthiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU)and analogs, streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) andgrowth factor inhibitors (e.g., fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin, mitoxantrone, topotecan, and irinotecan),corticosteroids (cortisone, dexamethasone, hydrocortisone,methylprednisolone, prednisone, and prednisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; and chromatin disruptors.

In some embodiments, a therapeutic agent comprises an immunotherapy.Cancer immunotherapy is the use of the immune system to reject cancer.The main premise is stimulating the subject's immune system to attackthe tumor cells that are responsible for the disease. This can be eitherthrough immunization of the subject, in which case the subject's ownimmune system is rendered to recognize tumor cells as targets to bedestroyed, or through the administration of therapeutics, such asantibodies, as drugs, in which case the subject's immune system isrecruited to destroy tumor cells by the therapeutic agents. Cancerimmunotherapy includes an antibody-based therapy and cytokine-basedtherapy.

A number of therapeutic monoclonal antibodies have been approved by theFDA for use in humans, and more are underway. The FDA-approvedmonoclonal antibodies for cancer immunotherapy include antibodiesagainst CD52, CD33, CD20, ErbB2, vascular endothelial growth factor andepidermal growth factor receptor. Examples of monoclonal antibodiesapproved by the FDA for cancer therapy include, without limitation:Rituximab (available as Rituxan™), Trastuzumab (available asHerceptin™), Alemtuzumab (available as Campath-IH™), Cetuximab(available as Erbitux™), Bevacizumab (available as Avastin™) Panitumumab(available as Vectibix™), Gemtuzumab ozogamicin (available as Mylotarg™)Ibritumomab tiuxetan (available as Zevalin™), Tositumomab (available asBexxar™) Ipilimumab (available as Yervoy™), Ofatunumab (available asArzerra™), Daclizumab (available as Zinbryta™), Nivolumab (available asOpdivo™), and Pembrolizumab (available as Keytruda™). Examples ofmonoclonal antibodies currently undergoing human clinical testing forcancer therapy in the United States include, without limitation: WX-G250(available as Rencarex™), Zanolimumab (available as HuMax-CD4), ch14.18,Zalutumumab (available as HuMax-EGFr), Oregovomab (available as B43.13,OvalRex™), Edrecolomab (available as IGN-101, Panorex™), 131I-chTNT-I/B(available as Cotara™), Pemtumomab (available as R-1549, Theragyn™),Lintuzumab (available as SGN-33), Labetuzumab (available as hMN14,CEAcide™), Catumaxomab (available as Removab™), CNTO 328 (available ascCLB8), 3F8, 177Lu-J591, Nimotuzumab, SGN-30, Ticilimumab (available asCP-675206), Epratuzumab (available as hLL2, LymphoCide™),90Y-Epratuzumab, Galiximab (available as IDEC-114), MDX-060, CT-011,CS-1008, SGN-40, Mapatumumab (available as TRM-I), Apolizumab (availableas HuID10, Remitogen™) and Volociximab (available as M200).

Cancer immunotherapy also includes a cytokine-based therapy. Thecytokine-based cancer therapy utilizes one or more cytokines thatmodulate a subject's immune response. Non-limiting examples of cytokinesuseful in cancer treatment include interferon-α (IFN-α), interleukin-2(IL-2), Granulocyte-macrophage colony-stimulating factor (GM-CSF) andinterleukin-12 (IL-12).

The B cell receptor ligand coupled to therapeutic agents, as well asencoding nucleic acids or nucleic acid sets, vectors comprising such, orhost cells comprising the vectors, described herein are useful fortreating cancer, including B cell malignancies, e.g. B cell lymphomas.

In some embodiments, more than one B cell receptor ligand coupled to atherapeutic agent, or a combination of a B cell receptor ligand coupledto a therapeutic agent and another suitable therapeutic agent, may beadministered to a subject in need of the treatment. The B cell receptorligand coupled to a therapeutic agent can also be used in conjunctionwith other agents that serve to enhance and/or complement theeffectiveness of the agents.

Also contemplated herein are methods of treatment comprisesconcomitantly administering CAR-expressing T-cells, wherein the CARcomprises an antigen binding domain that specifically binds acancer-specific antigen in a cancer-specific manner; and a vaccinecomprising a polypeptide or a nucleic acid expressing thecancer-specific antigen, or a cancer-specific fragment thereof. In someembodiments, the cancer-specific antigen comprises a B cell receptor andthe antigen binding domain comprises a B cell receptor ligand describedherein. In some embodiments, the antigen binding domain comprises a Bcell receptor ligand described herein identified by the methodsdescribed herein.

The terms “cancer-specific antigen” or “tumor antigen” interchangeablyrefers to a molecule (typically a protein, carbohydrate or lipid) thatis expressed on the surface of a cancer cell, either entirely or as afragment (e.g., MHC/peptide), and which is useful for the preferentialtargeting of a pharmacological agent to the cancer cell. In someembodiments, the cancer-specific antigen comprises a B cell receptor andthe antigen binding domain comprises a B cell receptor ligand describedherein. In some embodiments, the antigen binding domain comprises a Bcell receptor ligand described herein identified by the methodsdescribed herein. In some embodiments, a tumor antigen is a markerexpressed by both normal cells and cancer cells, e.g., a lineage marker,e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cellsurface molecule that is overexpressed in a cancer cell in comparison toa normal cell, for instance, 1-fold over expression, 2-foldoverexpression, 3-fold overexpression or more in comparison to a normalcell. In some embodiments, a tumor antigen comprises a somatic mutation,e.g., is a cell surface molecule that is inappropriately synthesized inthe cancer cell, for instance, a molecule that contains deletions,additions or mutations in comparison to the molecule expressed on anormal cell. In some embodiments, a cancer-specific antigen comprises apoint mutation, a splice-site mutation, a frameshift mutation, aread-through mutation, or a gene-fusion mutation. In some embodiments, acancer-specific antigen comprises a mutation in EGFRvIII, PSCA, BCMA,CD30, CEA, CD22, L1CAM, ROR1, ErbB, CD123, IL13Ra2, Mesothelin, FRα,VEGFR, c-Met, 5T4, CD44v6, B7-H4, CD133, CD138, CD33, CD28, GPC3, EphA2,CD19, ACVR2B, anaplastic lymphoma kinase (ALK), MYCN, BCR, HER2,NY-ESO1, MUC1, or MUC16. In some embodiments, a tumor antigen will beexpressed exclusively on the cell surface of a cancer cell, entirely oras a fragment (e.g., MHC/peptide), and not synthesized or expressed onthe surface of a normal cell.

In some embodiments, the cancer-specific antigen binds a cancer-specificantigen in a cancer-specific manner. In some embodiments, when a thecancer-specific antigen binds a cancer-specific antigen in acancer-specific manner, the cancer-specific antigen binds cancerouscells with 1.1×, 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×,40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, 300×, 400×, 500×, 600×, 700×,800×, 900×, 1,000× or more affinity than non-cancerous cells.

In some embodiments, the methods described herein comprise identifyingthe cancer-specific antigen in a subject. In some embodiments,identifying the cancer-specific antigen comprises obtaining cancerouscells from a subject. In some embodiments, the cancerous cells areobtained from a biopsy. In some embodiments, the cancerous cells are inthe blood of the subject.

In some embodiments, DNA from the cancerous cells is extracted andsequenced. In some embodiments, the sequence of the DNA, or of one ormore genes is compared to the same sequence in non-cancerous cells.

In some embodiments, RNA from the cancerous cells is extracted and cDNAis synthesized. In some embodiments, the cDNA is sequenced, In someembodiments, the sequence of the cDNA, or of one or more genes iscompared to the same sequence in non-cancerous cells.

In some embodiments, identifying the cancer-specific antigen comprisesisolating and sequencing circulating cell free DNA of the subject.

“Concomitantly” means administering two or more substances to a subjectin a manner that is correlated in time, preferably sufficientlycorrelated in time so as to provide a modulation in an immune response.In embodiments, concomitant administration may occur throughadministration of two or more substances in the same dosage form. Inother embodiments, concomitant administration may encompassadministration of two or more substances in different dosage forms, butwithin a specified period of time, preferably within 1 month, morepreferably within 1 week, still more preferably within 1 day, and evenmore preferably within 1 hour. The use of the term “concomitantly” doesnot restrict the order in which the therapeutic agents are administeredto a subject. A first therapeutic agent, such as a CAR-T cell, can beadministered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), simultaneously with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapeutic agent, suchas a vaccine described herein, to a subject. Thus, a first agent can beadministered separately, sequentially or simultaneously with the secondtherapeutic agent. In some embodiments, the concomitant administrationoccurs at least two times, at least three times, at least four times, atleast five times, at least six times, at least seven times, at leasteight times, at least nine times, or at least ten times in the subject.

In some embodiments, the CAR-expressing T cells are administered beforethe vaccine. In some embodiments, the CAR-expressing T cells areadministered after the vaccine.

To practice the method disclosed herein, an effective amount of the Bcell receptor ligand coupled to a therapeutic agent, the CARs, and thevaccines described herein can be administered to a subject (e.g., ahuman) in need of the treatment via a suitable route, such asintravenous administration, e.g., as a bolus or by continuous infusionover a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, inhalation or topical routes. In some embodiments,vaccines described herein are administered intratumorally. In someembodiments, CAR T-cells described herein are administeredintraveneously. Commercially available nebulizers for liquidformulations, including jet nebulizers and ultrasonic nebulizers areuseful for administration. Liquid formulations can be directly nebulizedand lyophilized powder can be nebulized after reconstitution.Alternatively, the B cell receptor ligand coupled to a therapeuticagent, the CARs, and the vaccines as described herein can be aerosolizedusing a fluorocarbon formulation and a metered dose inhaler, or inhaledas a lyophilized and milled powder.

The subject to be treated by the methods described herein can be amammal, more preferably a human. Mammals include, but are not limitedto, farm animals, sport animals, pets, primates, horses, dogs, cats,mice and rats. A human subject who needs the treatment may be a humanpatient having, at risk for, or suspected of having a targetdisease/disorder, such as cancer. A subject having a target disease ordisorder can be identified by routine medical examination, e.g.,laboratory tests, organ functional tests, CT scans, or ultrasounds. Asubject suspected of having any of such target disease/disorder mightshow one or more symptoms of the disease/disorder. A subject at risk forthe disease/disorder can be a subject having one or more of the riskfactors for that disease/disorder.

The methods and compositions described herein may be used to treat anydisease or disorder associated with cancer. In some embodiments, thecancer is a B cell malignancy. In some embodiments, the cancer is alymphoma. In some embodiments, the cancer is selected from diffuse largeB-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-celllymphoma (MZL) or mucosa-associated lymphatic tissue lymphoma (MALT),chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL),Burkitt's lymphoma, lymphoplasmacytic lymphoma, nodal marginal zone Bcell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL),intravascular large B-cell lymphoma, primary effusion lymphoma,lymphomatoid granulomatosis, primary central nervous system lymphoma,ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-celllymphoma arising in HHV8-associated multicentric Castleman's disease,and B-cell lymphoma.

Other cancers include but are not limited to: Oral: buccal cavity, lip,tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: non-small cell lung cancer (NSCLC), small cell lungcancer, bronchogenic carcinoma (squamous cell or epidermoid,undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, larynx,adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or smallintestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi'ssarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), largebowel or large intestines (adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma), rectal, colon, colon-rectum, colorectal;Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), head and neck cancer, meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastomamultiform, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), spinal cord neurofibroma, meningioma, glioma, sarcoma);Gynecological: uterus (endometrial carcinoma), cervix (cervicalcarcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma[serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoll-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast;Hematologic: blood (myeloid leukemia [acute and chronic], acutelymphoblastic leukemia, myeloproliferative diseases, multiple myeloma,myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma[malignant lymphoma] hairy cell; lymphoid disorders; Skin: malignantmelanoma, basal cell carcinoma, squamous cell carcinoma, Karposi'ssarcoma, keratoacanthoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis, Thyroid gland: papillary thyroidcarcinoma, follicular thyroid carcinoma; medullary thyroid carcinoma,multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma;and Adrenal glands: neuroblastoma.

As used herein, “an effective amount” refers to the amount of eachactive agent required to confer therapeutic effect on the subject,either alone or in combination with one or more other active agents. Insome embodiments, the therapeutic effect is reduction in progression ofcancer. Determination of whether an amount of the B cell receptor ligandcoupled to a therapeutic agent described herein, or the CARs and thevaccines described herein achieved the therapeutic effect would beevident to one of skill in the art. Effective amounts vary, asrecognized by those skilled in the art, depending on the particularcondition being treated, the severity of the condition, the individualpatient parameters including age, physical condition, size, gender andweight, the duration of the treatment, the nature of concurrent therapy(if any), the specific route of administration and like factors withinthe knowledge and expertise of the health practitioner. These factorsare well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is generallypreferred that a maximum dose of the individual components orcombinations thereof be used, that is, the highest safe dose accordingto sound medical judgment.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. Frequency ofadministration may be determined and adjusted over the course oftherapy, and is generally, but not necessarily, based on treatmentand/or suppression and/or amelioration and/or delay of a targetdisease/disorder.

In one example, dosages may be determined empirically in individuals whohave been given one or more administration(s) of the molecule.Individuals are given incremental dosages of the molecule. To assessefficacy of the B cell receptor ligand coupled to a therapeutic agent,or the CARs and the vaccines an indicator of the disease/disorder can befollowed.

For the purpose of the present disclosure, the appropriate dosage willdepend on the type and severity of the disease/disorder, whether the Bcell receptor ligand coupled to a therapeutic agent or the CARs and thevaccines described herein is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the B cell receptor ligand coupled to a therapeutic agent or the CARsand the vaccines, and the discretion of the attending physician.Typically the clinician will administer the B cell receptor ligandcoupled to a therapeutic agent or the CARs and the vaccines, until adosage is reached that achieves the desired result. In some embodiments,the desired result is a decrease the severity of cancer. Methods ofdetermining whether a dosage resulted in the desired result would beevident to one of skill in the art. Administration of one or more B cellreceptor ligands coupled to a therapeutic agents or the CARs and thevaccines can be continuous or intermittent, depending, for example, uponthe recipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of a B cell receptor ligandcoupled to a therapeutic agent or the CARs and the vaccines may beessentially continuous over a preselected period of time or may be in aseries of spaced dose, e.g., either before, during, or after developinga target disease or disorder.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has a target disease or disorder, a symptom of thedisease/disorder, or a predisposition toward the disease/disorder, withthe purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect the disorder, the symptom of the disease,or the predisposition toward the disease or disorder.

Alleviating a target disease/disorder includes delaying the developmentor progression of the disease, or reducing disease severity. Alleviatingthe disease does not necessarily require curative results. As usedtherein, “delaying” the development of a target disease or disordermeans to defer, hinder, slow, retard, stabilize, and/or postponeprogression of the disease. This delay can be of varying lengths oftime, depending on the history of the disease and/or individuals beingtreated. A method that “delays” or alleviates the development of adisease, or delays the onset of the disease, is a method that reducesprobability of developing one or more symptoms of the disease in a giventime frame and/or reduces extent of the symptoms in a given time frame,when compared to not using the method. Such comparisons are typicallybased on clinical studies, using a number of subjects sufficient to givea statistically significant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of a target disease or disorder includes initialonset and/or recurrence.

The B cell receptor ligand coupled to a therapeutic agent or the CARsand the vaccines described herein can be administered via conventionalroutes, e.g., administered orally, parenterally, by inhalation spray,topically, rectally, nasally, buccally, vaginally or via an implantedreservoir. The term “parenteral” as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intraarticular,intraarterial, intrasynovial, intrasternal, intrathecal, intralesional,and intracranial injection or infusion techniques. In addition, it canbe administered to the subject via injectable depot routes ofadministration such as using 1-, 3-, or 6-month depot injectable orbiodegradable materials and methods. In some examples, thepharmaceutical composition is administered intraocularly orintravitreally.

In one embodiment, the B cell receptor ligand coupled to a therapeuticagent or the CARs and the vaccines described herein is administered viasite-specific or targeted local delivery techniques. Examples ofsite-specific or targeted local delivery techniques include variousimplantable depot sources of the antibody or local delivery catheters,such as infusion catheters, an indwelling catheter, or a needlecatheter, synthetic grafts, adventitial wraps, shunts and stents orother implantable devices, site specific carriers, direct injection, ordirect application. See, e.g., PCT Publication No. WO 00/53211 and U.S.Pat. No. 5,981,568.

Targeted delivery of therapeutic compositions containing an antisensepolynucleotide, expression vector, or subgenomic polynucleotides canalso be used. Receptor-mediated DNA delivery techniques are describedin, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiouet al., Gene Therapeutics: Methods And Applications Of Direct GeneTransfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988)263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc.Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991)266:338.

The therapeutic polynucleotides and polypeptides described herein can bedelivered using gene delivery vehicles. The gene delivery vehicle can beof viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy(1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, HumanGene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148).Expression of such coding sequences can be induced using endogenousmammalian or heterologous promoters and/or enhancers. Expression of thecoding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.Additional approaches are described in Philip, Mol. Cell. Biol. (1994)14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

The particular dosage regimen, i.e., dose, timing and repetition, usedin the method described herein will depend on the particular subject andthat subject's medical history.

Treatment efficacy for a target disease/disorder can be assessed bymethods well-known in the art.

As used herein, the term “in combination” refers to the use of more thanone therapeutic agent. The use of the term “in combination” does notrestrict the order in which the therapeutic agents are administered to asubject. A first therapeutic agent can be administered prior to (e.g., 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapeutic agent. Thus, a first agent can be administeredseparately, sequentially or simultaneously with the second therapeuticagent.

In some embodiments, a CAR-T cell and a vaccine described herein areadministered in combination with a TLR9 agonist. In some embodiments,the TLR9 agonist is a CpG oligonucleotide.

Vaccines

In some embodiments, CAR-expressing T-cells described herein areadministered with a vaccine. In some embodiments, the vaccine comprisesa polypeptide or a nucleic acid expressing a cancer-specific antigen, ora cancer-specific fragment thereof, as is described supra.

In some embodiments, the vaccine comprises a cancer-specific fragment ofa cancer-specific antigen.

In some embodiments, the cancer-specific fragment of the cancer specificantigen is 1-1000 amino acids long, or 10-500 amino acids long. In someembodiments, the cancer-specific fragment of the cancer specific antigenis 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 ormore amino acids long.

In some embodiments, the cancer-specific antigen, or a cancer-specificfragment thereof comprises a somatic mutation as is described supra,e.g., comprises a point mutation, a splice-site mutation, a frameshiftmutation, a read-through mutation, or a gene-fusion mutation, and thepolypeptide or nucleic acid expressing the cancer-specific antigen, orcancer-specific fragment thereof comprises the somatic mutation.

Peptide Vaccines

In some embodiments, the vaccine comprises a polypeptide expressing acancer-specific antigen, or a cancer-specific fragment thereof.

In particular embodiments, the DNA that encodes for the protein vaccinecan be introduced into an expression vector, such as a plasmid. Multiplecloning sites, which contain DNA sequences that are recognized byrestriction enzymes, can facilitate the insertion of the protein vaccineDNA into the vector. In particular embodiments, DNA constructs (such asexpression vectors) that encode the proteins of interest can beintroduced into cells to induce protein expression and the cells can beharvested to extract the protein of interest. The DNA encoding theprotein of interest can be included in an expression vector that alsocontains sequences that control gene expression, such as promotersequences. 5′ and 3′ untranslated regions can be encoded upstream anddownstream of the protein coding sequence in order to enhanceexpression. For example, a 5′ untranslated leader sequence and a 3′polyadenylation sequence can be used. In particular embodiments, the DNAcan be introduced into cells for protein expression by heat-shocktransformation. In particular embodiments, DNA can be introduced intocells for protein expression by transfection, electroporation,impalefection or hydrodynamic delivery. In particular embodiments, theDNA used for protein expression can be delivered in the form of a viralvector. In particular embodiments, the protein of interest can beharvested from lysed cells, and purified. Protein purification can beperformed using size-exclusion chromatography, or by a chromatographytechnique that isolates the protein based on a protein-tag, such as a 6×histidine tag or a c-myc tag. The histidine tag can be encoded adjacentto a sequence recognized and cleaved by a protease, to facilitateremoval of the histidine tag after protein purification. An example of aprotease that can be used to remove a histidine tag from a protein isthe human rhinovirus 3C protease.

In some embodiments, the vaccine comprises two or more polypeptideshaving overlapping sequences, each expressing a fragment of thecancer-specific antigen.

In some embodiments, the polypeptide is conjugated to a carrier protein,e.g., OVA, KLH, or BSA.

DNA Vaccines

In some embodiments, the vaccine comprises a nucleic acid expressing acancer-specific antigen, or a cancer-specific fragment thereof.

In some embodiments, the nucleic acid is DNA. A DNA vaccine may comprisean “expression vector” or “expression cassette,” i.e., a nucleotidesequence which is capable of affecting expression of a protein codingsequence in a host compatible with such sequences. Expression cassettesinclude at least a promoter operably linked with the polypeptide codingsequence; and, optionally, with other sequences, e.g., transcriptiontermination signals. Additional factors necessary or helpful ineffecting expression may also be included, e.g., enhancers.

“Operably linked” means that the coding sequence is linked to aregulatory sequence in a manner that allows expression of the codingsequence. Known regulatory sequences are selected to direct expressionof the desired protein in an appropriate host cell. Accordingly, theterm “regulatory sequence” includes promoters, enhancers and otherexpression control elements. Such regulatory sequences are described in,for example, Goeddel, Gene Expression Technology. Methods in Enzymology,vol. 185, Academic Press, San Diego, Calif. (1990)).

A promoter region of a DNA or RNA molecule binds RNA polymerase andpromotes the transcription of an “operably linked” nucleic acidsequence. As used herein, a “promoter sequence” is the nucleotidesequence of the promoter which is found on that strand of the DNA or RNAwhich is transcribed by the RNA polymerase. Two sequences of a nucleicacid molecule, such as a promoter and a coding sequence, are “operablylinked” when they are linked to each other in a manner which permitsboth sequences to be transcribed onto the same RNA transcript or permitsan RNA transcript begun in one sequence to be extended into the secondsequence. Thus, two sequences, such as a promoter sequence and a codingsequence of DNA or RNA are operably linked if transcription commencingin the promoter sequence will produce an RNA transcript of the operablylinked coding sequence. In order to be “operably linked” it is notnecessary that two sequences be immediately adjacent to one another inthe linear sequence.

The preferred promoter sequences of the present invention must beoperable in mammalian cells and may be either eukaryotic or viralpromoters. Suitable promoters may be inducible, repressible orconstitutive. A “constitutive” promoter is one which is active undermost conditions encountered in the cell's environmental and throughoutdevelopment. An “inducible” promoter is one which is under environmentalor developmental regulation. A “tissue specific” promoter is active incertain tissue types of an organism. An example of a constitutivepromoter is the viral promoter MSV-LTR, which is efficient and active ina variety of cell types, and, in contrast to most other promoters, hasthe same enhancing activity in arrested and growing cells. Otherpreferred viral promoters include that present in the CMV-LTR (fromcytomegalovirus) (Bashart, M. et al., Cell 41:521, 1985) or in theRSV-LTR (from Rous sarcoma virus) (Gorman. C. M., Proc. Natl. Acad. Sci.USA 79:6777, 1982). Also useful are the promoter of the mousemetallothionein I gene (Hamer. D, et al., J. Mol. Appl. Gen. 1:273-88,1982; the TK promoter of Herpes virus (McKnight. S, Cell 31:355-65,1982): the SV40 early promoter (Benoist. C., et al., Nature 290:304-10,1981): and the yeast gal4 gene promoter (Johnston, S A et al., Proc.Natl. Acad. Sci. USA 79:6971-5, 1982); Silver, P A. et al., Proc. Natl.Acad. Sci. (USA) 81:5951-5, 1984)). Other illustrative descriptions oftranscriptional factor association with promoter regions and theseparate activation and DNA binding of transcription factors include:Keegan et al., Nature 231:699, 1986: Fields et al., Nature 340:245,1989; Jones, Cell 61:9, 1990; Lewin. Cell 61:1161, 1990: Ptashne et al.,Nature 346:329, 1990; Adams et al., Cell 72:306, 1993.

The promoter region may further include an octamer region which may alsofunction as a tissue specific enhancer, by interacting with certainproteins found in the specific tissue. The enhancer domain of the DNAconstruct of the present invention is one which is specific for thetarget cells to be transfected, or is highly activated by cellularfactors of such target cells. Examples of vectors (plasmid orretrovirus) are disclosed, e.g., in Roy-Burman et al., U.S. Pat. No.5,112,767. For a general discussion of enhancers and their actions intranscription, see. Lewin, B M, Genes IV. Oxford University Press pp.552-576, 1990 (or later edition). Particularly useful are retroviralenhancers (e.g., viral LTR) that is preferably placed upstream from thepromoter with which it interacts to stimulate gene expression. For usewith retroviral vectors, the endogenous viral LTR may be renderedenhancer-less and substituted with other desired enhancer sequenceswhich confer tissue specificity or other desirable properties such astranscriptional efficiency.

Thus, expression cassettes include plasmids, recombinant viruses, anyform of a recombinant “naked DNA” vector, and the like. A “vector”comprises a nucleic acid which can infect, transfect, transiently orpermanently transduce a cell. It will be recognized that a vector can bea naked nucleic acid, or a nucleic acid complexed with protein or lipid.The vector optionally comprises viral or bacterial nucleic acids and/orproteins, and/or membranes (e.g., a cell membrane, a viral lipidenvelope, etc.). Vectors include replicons (e.g., RNA replicons),bacteriophages) to which fragments of DNA may be attached and becomereplicated. Vectors thus include, but are not limited to RNA, autonomousself-replicating circular or linear DNA or RNA, e.g., plasmids, viruses,and the like (U.S. Pat. No. 5,217,879), and includes both the expressionand nonexpression plasmids. Where a recombinant cell or culture isdescribed as hosting an “expression vector” this includes bothextrachromosomal circular and linear DNA and DNA that has beenincorporated into the host chromosome(s). Where a vector is beingmaintained by a host cell, the vector may either be stably replicated bythe cells during mitosis as an autonomous structure, or is incorporatedwithin the host's genome.

Exemplary virus vectors that may be used include recombinantadenoviruses (Horowitz, M S, In: Virology. Fields, B N et al., eds.Raven Press, NY, 1990, p. 1679; Berkner, K L. Biotechniques 6:616-29,1988; Strauss. S E. In: The Adenoviruses, Ginsberg, H S, ed., PlenumPress, N Y, 1984, chapter 11) and herpes simplex virus (HSV). Advantagesof adenovirus vectors for human gene delivery include the fact thatrecombination is rare, no human malignancies are known to be associatedwith such viruses, the adenovirus genome is double stranded DNA whichcan be manipulated to accept foreign genes of up to 7.5 kb in size, andlive adenovirus is a safe human vaccine organisms. Adeno-associatedvirus is also useful for human therapy (Samulski. R J et al., EMBO J.10:3941, 1991) according to the present invention.

Another vector which can express the DNA molecule of the presentinvention, and is useful in the present therapeutic setting is vacciniavirus, which can be rendered non-replicating (U.S. Pat. Nos. 5,225,336;5.204,243; 5,155,020; 4,769,330; Fuerst, T R et al., Proc. Natl. Acad.Sci. USA 86:2549-53, 1992; Chakrabarti, S et al., Mol Cell Biol5:3403-9, 1985). Descriptions of recombinant vaccinia viruses and otherviruses containing heterologous DNA and their uses in immunization andDNA therapy are reviewed in: Moss, B, Curr Opin Genet Dev 3:86-90.1993;Moss, B, Biotechnol, 20:345-62, 1992).

Other viral vectors that may be used include viral or non-viral vectors,including adeno-associated virus vectors, retrovirus vectors, lentivirusvectors, and plasmid vectors. Exemplary types of viruses include HSV(herpes simplex virus), AAV (adeno associated virus), HIV (humanimmunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV(murine leukemia virus).

A DNA vaccine may also use a replicon, e.g., an RNA replicon, aself-replicating RNA vector. Generally, RNA replicon vaccines may bederived from alphavirus vectors, such as Sindbis virus (Hariharan, M Jet al., 1998. J Virol 72:950-8), Semliki Forest virus (Berglund, P M etal., 1997. AIDS Res Hum Retroviruses 13:1487-95; Ying, H T et al., 1999.Nat Med 5:823-7) or Venezuelan equine encephalitis virus (Pushko, P M etal., 1997. Virology 239:389-401). These self-replicating andself-limiting vaccines may be administered as either (1) RNA or (2) DNAwhich is then transcribed into RNA replicons in cells transfected invitro or in vivo (Berglund, P C et al., 1998. Nat Biotechnol 16:562-5;Leitner, W W et al., 2000. Cancer Res 60:51-5). An exemplary SemlikiForest virus is pSCA1 (DiCiommo, D P et al., J Biol Chem 1998;273:18060-6).

In addition to naked DNA or viral vectors, engineered bacteria may beused as vectors. A number of bacterial strains including Salmonella, BCGand Listeria monocytogenes(LM) (Hoiseth et al., Nature 291:238-9, 1981;Poirier, T P et al., J Exp Med 168:25-32, 1988); Sadoff. J C et al.,Science 240:336-8, 1988; Stover. C K et al., Nature 351:456-60, 1991;Aldovini, A et al., Nature 351:479-82, 1991). These organisms displaytwo promising characteristics for use as vaccine vectors: (1) entericroutes of infection, providing the possibility of oral vaccine delivery;and (2) infection of monocytes/macrophages thereby targeting antigens toprofessional APCs.

In addition to virus-mediated gene transfer in vivo, physical meanswell-known in the art can be used for direct transfer of DNA, includingadministration of plasmid DNA (Wolff et al., 1990, supra) andparticle-bombardment mediated gene transfer (Yang, N-S. et al., ProcNatl Acad Sci USA 87:9568, 1990; Williams. R S et al., Proc Natl AcadSci USA 88:2726, 1991; Zelenin, A V et al., FEBS Lett 280:94, 1991;Zelenin, A V et al., FEBS Lett 244:65, 1989); Johnston, S A et al., InVitro Cell Dev Biol 27:11, 1991). Furthermore, electroporation, awell-known means to transfer genes into cell in vitro, can be used totransfer DNA molecules according to the present invention to tissues invivo (Titomirov. A V et al., Biochim Biophys Acta 1088:131, 1991).

“Carrier mediated gene transfer” has also been described (Wu, C H etal., J Biol Chem 264:16985, 1989; Wu, G Y et al., J Biol Chem 263:14621,1988; Soriano, P et al., Proc Nat. Acad Sci USA 80:7128, 1983; Wang. C-Yet al., Pro. Natl Acad Sci USA 84:7851, 1982; Wilson, J M et al., J BiolChem 267:963, 1992). Preferred carriers are targeted liposomes (Nicolau,C et al., Proc Natl Acad Sci USA 80:1068, 1983; Soriano et al., supra)such as immunoliposomes, which can incorporate acylated mAbs into thelipid bilayer (Wang et al., supra). Polycations such asasialoglycoprotein/polylysine (Wu et al., 1989, supra) may be used,where the conjugate includes a target tissue-recognizing molecule (e.g.,asialo-orosomucoid for liver) and a DNA binding compound to bind to theDNA to be transfected without causing damage, such as polylysine. Thisconjugate is then complexed with plasmid DNA of the present invention.

Plasmid DNA used for transfection or microinjection may be preparedusing methods well-known in the art, for example using the Qiagenprocedure (Qiagen), followed by DNA purification using known methods,such as the methods exemplified herein.

Such expression vectors may be used to transfect host cells (in vitro,ex vivo or in vivo) for expression of the DNA and production of theencoded proteins which include fusion proteins or peptides. In oneembodiment, a DNA vaccine is administered to or contacted with a cell.e.g., a cell obtained from a subject (e.g., an antigen presenting cell),and administered to a subject, wherein the subject is treated before,after or at the same time as the cells are administered to the subject.

RNA Vaccines

In some embodiments, the vaccine comprises a nucleic acid expressing acancer-specific antigen, or a cancer-specific fragment thereof, and thenucleic acid is RNA.

RNA vaccines, as provided herein, comprise at least one (one or more)ribonucleic acid (RNA) polynucleotide having an open reading frameencoding at least one cancer-specific antigen, or a cancer-specificfragment thereof. The term “nucleic acid,” in its broadest sense,includes any compound and/or substance that comprises a polymer ofnucleotides. These polymers are referred to as polynucleotides.

In some embodiments, polynucleotides of the present disclosure functionas messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to anypolynucleotide that encodes a (at least one) polypeptide (anaturally-occurring, non-naturally-occurring, or modified polymer ofamino acids) and can be translated to produce the encoded polypeptide invitro, in vivo, in situ or ex vivo.

The basic components of an mRNA molecule typically include at least onecoding region, a 5′ untranslated region (UTR), a 3′ UTR, a 5′ cap and apoly-A tail. Polynucleotides of the present disclosure may function asmRNA but can be distinguished from wild-type mRNA in their functionaland/or structural design features which serve to overcome existingproblems of effective polypeptide expression using nucleic-acid basedtherapeutics.

RNA (e.g., mRNA) vaccines of the present disclosure comprise, in someembodiments, at least one ribonucleic acid (RNA) polynucleotide havingan open reading frame encoding at least one a cancer-specific antigen,or a cancer-specific fragment thereof, wherein said RNA comprises atleast one chemical modification.

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise various (more than one)different modifications. In some embodiments, a particular region of apolynucleotide contains one, two or more (optionally different)nucleoside or nucleotide modifications. In some embodiments, a modifiedRNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced toa cell or organism, exhibits reduced degradation in the cell ororganism, respectively, relative to an unmodified polynucleotide. Insome embodiments, a modified RNA polynucleotide (e.g., a modified mRNApolynucleotide), introduced into a cell or organism, may exhibit reducedimmunogenicity in the cell or organism, respectively (e.g., a reducedinnate response).

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise non-natural modifiednucleotides that are introduced during synthesis or post-synthesis ofthe polynucleotides to achieve desired functions or properties. Themodifications may be present on an internucleotide linkages, purine orpyrimidine bases, or sugars. The modification may be introduced withchemical synthesis or with a polymerase enzyme at the terminal of achain or anywhere else in the chain. Any of the regions of apolynucleotide may be chemically modified.

The present disclosure provides for modified nucleosides and nucleotidesof a polynucleotide (e.g., RNA polynucleotides, such as mRNApolynucleotides). A “nucleoside” refers to a compound containing a sugarmolecule (e.g., a pentose or ribose) or a derivative thereof incombination with an organic base (e.g., a purine or pyrimidine) or aderivative thereof (also referred to herein as “nucleobase”). Anucleotide” refers to a nucleoside, including a phosphate group.Modified nucleotides may by synthesized by any useful method, such as,for example, chemically, enzymatically, or recombinantly, to include oneor more modified or non-natural nucleosides. Polynucleotides maycomprise a region or regions of linked nucleosides. Such regions mayhave variable backbone linkages. The linkages may be standardphosphdioester linkages, in which case the polynucleotides wouldcomprise regions of nucleotides.

Cancer vaccines of the present disclosure comprise at least one RNApolynucleotide, such as a mRNA (e.g., modified mRNA). mRNA, for example,is transcribed in vitro from template DNA, referred to as an “in vitrotranscription template.” In some embodiments, an in vitro transcriptiontemplate encodes a 5′ untranslated (UTR) region, contains an openreading frame, and encodes a 3′ UTR and a polyA tail. The particularnucleic acid sequence composition and length of an in vitrotranscription template will depend on the mRNA encoded by the template.

In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides.For example, a polynucleotide may include 200 to 500, 200 to 1000, 200to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to3000 nucleotides).

In other aspects, the invention relates to a method for preparing anmRNA cancer vaccine by IVT methods. In vitro transcription (IVT) methodspermit template-directed synthesis of RNA molecules of almost anysequence. The size of the RNA molecules that can be synthesized usingIVT methods range from short oligonucleotides to long nucleic acidpolymers of several thousand bases. IVT methods permit synthesis oflarge quantities of RNA transcript (e.g., from microgram to milligramquantities) (Beckert et al., Synthesis of RNA by in vitro transcription,Methods Mol Biol. 703:29-41(2011); Rio et al. RNA: A Laboratory Manual.Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220;Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed. WashingtonD.C.: ASM Press, 2007.262-299). Generally, IVT utilizes a DNA templatefeaturing a promoter sequence upstream of a sequence of interest. Thepromoter sequence is most commonly of bacteriophage origin (ex, the T7,T3 or SP6 promoter sequence) but many other promotor sequences can betolerated including those designed de novo. Transcription of the DNAtemplate is typically best achieved by using the RNA polymerasecorresponding to the specific bacteriophage promoter sequence. ExemplaryRNA polymerases include, but are not limited to T7 RNA polymerase, T3RNA polymerase, or SP6 RNA polymerase, among others. IVT is generallyinitiated at a dsDNA but can proceed on a single strand.

Vaccine Compositions

In some embodiments, the vaccine minimally includes the antigen.

To further achieve an effective vaccine according to this disclosure,materials and methods can be employed to enhance availability of thevaccine. One such method employs an adjuvant.

The term “adjuvant” refers to material that enhances the immune responseto an antigen and is used herein in the customary use of the term. Theprecise mode of action is not understood for all adjuvants, but suchlack of understanding does not prevent their clinical use for a widevariety of vaccines, whether protein-based or DNA-based. Traditionally,some adjuvants physically trap antigen at the site of injection,enhancing antigen presence at the site and slowing its release. This inturn prolongs and/or increases the recruitment and activation of APCs,such as in this case iDCs.

In particular embodiments a squalene-based adjuvant is used. Squalene ispart of the group of molecules known as triterpenes, which are allhydrocarbons with 30 carbon molecules. Squalene can be derived fromcertain plant sources, such as rice bran, wheat germ, amaranth seeds,and olives, as well as from animal sources, such as shark liver oil. Inparticular embodiments, the squalene-based adjuvant is MF59®, which isan oil-in-water emulsion (Novartis, Basel, Switzerland; see Giudice, G Det al. Clin Vaccine Immunol. 2006 Sep; 13(9):1010-3). An example of asqualene-based adjuvant that is similar to MF59® but is designed forpreclinical research use is Addavax™ (InvivoGen, San Diego, Calif.).MF59 has been FDA approved for use in an influenza vaccine, and studiesindicate that it is safe for use during pregnancy (Tsai T, et al.Vaccine. 2010. 17:28(7):1877-80; Heikkinen T, et al. Am J ObstetGynecol. 2012.207(3):177). In particular embodiments, squalene-basedadjuvants can include 0.1%-20% (v/v) squalene oil. In particularembodiments, squalene-based adjuvants can include 5%(v/v) squalene oil.In particular embodiments, the squalene-based adjuvant is AS03, whichincludes α-tocopherol, squalene, and polysorbate 80 in an oil-in-wateremulsion (GlaxoSmithKline; see Garcon N et al. Expert Rev Vaccines. 2012March; 11(3):349-66).

In particular embodiments, polyinosinic:polycytidilyic acid (alsoreferred to as poly(I:C) is used. Poly(I:C) is a synthetic analog ofdouble-stranded RNA that stimulates the immune system. In particularembodiments, Poly-ICLC (Hiltinol) is used (Ammi R et al. Pharmacol Ther.2015 February; 146:120-31). In particular embodiments, Poliu-IC12U(Ampligen) is used (Martins K A et al. Expert Rev Vaccines. 2015 March;14(3):447-59).

In particular embodiments the adjuvant alum can be used. Alum refers toa family of salts that contain two sulfate groups, a monovalent cation,and a trivalent metal, such as aluminum or chromium. Alum is an FDAapproved adjuvant. In particular embodiments, vaccines can include alumin the amounts of 1-1000 ug/dose or 0.1 mg-10 mg/dose.

In particular embodiments, the adjuvant Vaxfectin® (Vical, Inc., SanDiego, Calif.) can be used. Vaxfectin® is a cationic lipid basedadjuvant that can be used for DNA or protein vaccines.

Compositions for Administration. Vaccines of the disclosure can beformulated into pharmaceutical compositions for administration includinga vaccine of the disclosure can be formulated in a variety of forms,e.g., as a liquid, gel, lyophilized, or as a compressed solid. Theparticular form will depend upon the particular indication being treatedand will be apparent to one of ordinary skill in the art.

An example of a pharmaceutical composition is a solution designed forparenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations can also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by those orordinary skill in the art that lyophilized preparations are generallymore stable than their liquid counterparts. Such lyophilizedpreparations are reconstituted prior to use by the addition of one ormore suitable pharmaceutically acceptable diluents such as sterile waterfor injection or sterile physiological saline solution.

Parenterals can be prepared for storage as lyophilized formulations oraqueous solutions by mixing, as appropriate, the composition having thedesired degree of purity with one or more pharmaceutically acceptablecarriers, excipients or stabilizers typically employed in the art (allof which are termed “excipients”), for example buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants and/or other miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from 2 mM to 50 mM. Suitable buffering agents for use with thepresent disclosure include both organic and inorganic acids and saltsthereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives can be added to retard microbial growth, and are typicallyadded in amounts of 0.2%-1% (w/v). Suitable exemplary preservatives foruse with the present disclosure include phenol, benzyl alcohol,meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., benzalkonium chloride,bromide or iodide), hexamethonium chloride, alkyl parabens such asmethyl or propyl paraben, catechol, resorcinol, cyclohexanol and3-pentanol.

Isotonicifiers can be added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Polyhydric alcohols can be present in an amount between 0.1%and 25% by weight, typically 1% to 5%, taking into account the relativeamounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thevaccine or helps to prevent denaturation or adherence to the containerwall. Typical stabilizers can be polyhydric sugar alcohols (enumeratedabove); amino acids such as arginine, lysine, glycine, glutamine,asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine,glutamic acid, threonine, etc., organic sugars or sugar alcohols, suchas lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol,myoinisitol, galactitol, and glycerol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol,alpha-monothioglycerol and sodium thiosulfate; low molecular weightpolypeptides (i.e., <10 residues); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; monosaccharides such as xylose, mannose,fructose and glucose; disaccharides such as lactose, maltose andsucrose; trisaccharides such as raffinose, and polysaccharides such asdextran. Stabilizers are typically present in the range of from 0.1 to10,000 parts by weight based on the vaccine composition. Additionalmiscellaneous excipients include bulking agents or fillers (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E) and cosolvents.

The vaccine composition can also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences.

Parenteral formulations to be used for in vivo administration generallyare sterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

Suitable examples of sustained-release vaccine compositions includesemi-permeable matrices of solid hydrophobic polymers containing thecomposition, the matrices having a suitable form such as a film ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the PROLEASE® (Alkermes,Inc., Waltham, Mass.) technology or LUPRON DEPOT® (injectablemicrospheres composed of lactic acid-glycolic acid copolymer andleuprolide acetate; Abbott Endocrine, Inc., Abbott Park, Ill.), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forlong periods, such as up to or over 100 days, certain hydrogels releasecompounds for shorter time periods.

EXAMPLES Example 1

Reported herein is the development of a novel platform to significantlyenhance the efficacy and safety of Follicular lymphoma treatment. Sincelymphoma is a clonal malignancy of a diversity system, every tumor has adifferent antibody on its cell surface. Combinatorial autocrine-basedselection is used to rapidly identify specific ligands for these B cellreceptors on the surface of FL tumor cells. The selected ligands areused in a CAR-T format for redirection of human CTLs. Essentially, theformat is the inverse of the usual CAR-T protocol. Instead of being aguide molecule, the antibody itself is the target. Thus, these studiesraise the possibility of personalized treatment of lymphomas utilizing aprivate antibody binding ligand that can be obtained in few weeks.

Although a special case, the B cell receptor (BCR) on lymphoma cells isthe purest form of a tumor specific antigen (1). This is becauselymphoma is a tumor of one member of a diversity system were each tumorexpresses only one of 10⁸ different antibody molecules (2). Thus, it'sremarkable that antigens selective for BCR's binding have not been moregenerally used for therapy (3, 4). Probably, the reason is that theworkflow to find a selective antigen for each patient is not possible inmost therapeutic settings. Here we describe an autocrine-based formatthat allows identification of peptide antigens selective for individualBCR's with a speed compatible with their use in the clinic. Theseselected antigens can be used as guide molecules for CAR-T or otherapproaches such as radiotherapy. The main point is that autocrine-basedselections allow for the speed and specificity that are required ifpersonalized therapy of lymphoma is to be realized.

Materials and Methods Identification and Reconstitution of LymphomaCells BCR

Lymph nodes biopsies from patients with follicular lymphoma diagnosis(FL) were kindly provided by N.N. Petrov Research Institute of Oncology(St. Petersburg, Russia).

Immediately after surgery the biopsy sample was separated to four equalslices, two of them were loaded into the RNAlater reagent (Qiagen) andothers were cryopreserved. Lymphoma cell counts and expression ofsurface Ig is determined by flow cytometry. Cell suspension aliquotscontaining approx. 250,000 cells were stained with monoclonal antibodiesin 4 tubes: 1. Isotype control; 2. CD45-FITC, CD20-PE, CD3-PC5,CD19-PE-Cy7; 3. IgG-PE-Cy5, IgM-FITC, CD19-PECy7; and 4. kappa-FITC,lambda-PE, CD19-PE-Cy7. Immunoglobulin expression was estimated onlymphocytes as gated using SSC/FSC and CD19C. Monoclonal immunoglobulinexpression of either M or G heavy chain, either kappa or lymbda lightchain was detected. The RNAlater processed biopsy samples were used forisolation of the total mRNA using RNAeasy Mini Kit (Qiagen). Total cDNAwas synthesized by reverse transcription using a QuantiTect ReverseTranscription Kit (Qiagen). Variable region genes of heavy and light Igchains identified by flow cytometry were amplified in separate reactionsfor each gene. Semi-nested PCR using high-fidelity DNA-polymerase (Q5,NEB) with a set of family specific V-gene forward primers and a C-genespecific reverse primer was used (Table 1). First step PCR products weresubjected to heteroduplex analysis in polyacrylamide gel to discriminatehomoduplexes (monoclonal PCR products) from a smear of slowly movingheteroduplexes (derived from polyclonal lymphocytes). DNA fragments ofthe expected size are extracted and the DNA eluted. Proximal reverseC-gene specific primer was used for the second step amplification andsequencing. Identified variable fragments of the follicular lymphomaBCRs were cloned as a scFv into the lentiviral vector pLV2-Fc-MTA codingfor a membrane-anchored human antibody Fc fragment (S) (FIGS. 5B and 5C)(FL). Jurkat and Raji cells were transduced with these viruses.Transduced Jurkat-FL and Raji-FL were analyzed by FACS in order toselect the cells carrying the follicular lymphoma BCR, which were thenused for autocrine selections or animal experiments.

TABLE 1 List of primers for variable region genes ofheavy and light Ig chains amplification. Primer Sequence 5′-3′Orientation L-VH1-start ATGGACTGGACCTGGAGGATC forward CT (SEQ ID NO: 4)L-VH2-start ATGGACATACTTTGTTCCACG forward CTC (SEQ ID NO: 5) L-VH3-startATGGAGTTTGGGCTGAGCTGG forward (SEQ ID NO: 6) L-VH4-startATGAAACACCTGTGGTTCTTC forward CT (SEQ ID NO: 7) L-VHS-startATGGGGTCAACCGCCATCCTC forward (SEQ ID NO: 8) L-VH6-startATGTCTGTCTCCTTCCTCATC forward TTC (SEQ ID NO: 9) IgM-3′CTCTCAGGACTGATGGGAAGC reverse C (SEQ ID NO: 10) distal IgM-clonGGAGACGAGGGGGAAAAG reverse (SEQ ID NO: 11) proximal IgG-3′GCCTGAGTTCCACGACACC reverse (SEQ ID NO: 12) distal IgG-clonCAGGGGGAAGACCGATGG reverse (SEQ ID NO: 13) proximal Vκ1-clonGACATCCAGATGACCCAGTCT forward CC (SEQ ID NO: 14) Vκ2-clonGATATTGTGATGACCCAGACT forward CCA (SEQ ID NO: 15) Vκ3-clonGAAATTGTGTTGACACAGTCT forward CCA (SEQ ID NO: 16) IGKC-3′CCCCTGTTGAAGCTCTTTGT reverse (SEQ ID NO: 17) distal IGKC-clonAGATGGCGGGAAGATGAAG reverse (SEQ ID NO: 18) proximal VL1_(51)_clonCAGTCTGTGTTGACGCAGCCG forward CCCTC (SEQ ID NO: 19) VL1_(36-47)_clonTCTGTGCTGACTCAGCCACCC forward TC (SEQ ID NO: 20) VL1_(40)_clonCAGTCTGTCGTGACGCAGCCG forward CCCTC (SEQ ID NO: 21) VL2-clonTCCGTGTCCGGGTCTCCTGGA forward CAGTC (SEQ ID NO: 22) VL3-clonACTCAGCCACCCTCGGTGTCA forward GTG (SEQ ID NO: 23) VL4-clonTCCTCTGCCTCTGCTTCCCTG forward GGA (SEQ ID NO: 24) VL5-clonCAGCCTGTGCTGACTCAGCC forward (SEQ ID NO: 25) IGLC-3′ GTGTGGCCTTGTTGGCTTGreverse (SEQ ID NO: 26) distal IGLC2-7_clon CGAGGGGGCAGCCTTGGG reverse(SEQ ID NO: 27) proximal IGLC1_clon AGTGACCGTGGGGTTGGCCTT reverseGGG (SEQ ID NO: 28) proximal

Construction of a CAR-Based Combinatorial Peptide Library

The DNA fragment coding for the 3rd generation chimeric antigen T-cellreceptor was synthesized (GeneCust) and cloned into the pLV2 lentiviralvector (Clontech) under control of the EF1a promoter. The arrangement ofgenes are in the order of: interleukin 2 signal peptide at the Nterminus; IgG1 Fc spacer domain with modified PELLGG and ISR motifs;GGGS linker; a CD28 trans-membrane and intracellular region;intracellular domains of the OX-40 and CD3zetta (FIGS. 5A and 5C). Toconstruct the combinatorial cyclopeptide library, randomized peptides inthe format of CX₇C, (X=20 natural amino acids) were appended to the Nterminus of the Fc domain by PCR using oligonucleotides with degenerateNNK codons. The diversity of the generated library was estimated as 109members. The lentiviral library of CX₇C-Fc-CAR was prepared byco-transfection of HEK293T cells with the library plasmid and thepackaging plasmids. Supernatants containing virus were collected at 48 hpost transfection. The titer of lentivirus preparations was determinedusing Lenti-X p24 ELISAs (Clontech).

FACS-Based Sorting

Jurkat-FL1, Jurkat-FL2 and Jurkat-FL3 cells were transduced with thelentiviral cyclopeptide-CAR library. Two days post-infection,CD69-positive cells were sorted using a FACSAria III (BD Biosciences).The peptides sequences were determined directly from sorted cells by PCRof the genes that encode them and were cloned into the lentiviral vectorto construct libraries for the next round of selection. Four iterativerounds of selection were carried out.

Cells and Culturing Conditions

Cell lines were cultured in media supplemented with 10% FBS (Gibco), 10mM HEPES, 100 U/ml penicillin, 100 ug/ml streptomycin, and 2 mM GlutaMAX(Gibco). The 293T lentiviral packaging cell line (Clontech) and HEp-2cell line were cultured in DMEM (Gibco). Human HEp-2 (CCL-23), Jurkat(TIB-152) and Raji (CCL-86) cell lines were obtained from the Instituteof Cytology RAS culture collection (St. Petersburg, Russia). The Jurkat,Jurkat-FL, Raji and Raji-FL cell lines were cultured in RPMI (Gibco).Human peripheral blood mononuclear cells (PBMCs) were isolated from theblood of healthy donors by gradient density centrifugation on aFicoll-Paque (GE Healthcare), washed and then re-suspended in serum-freeRPMI.

CD8⁺ T Cell Activation, Expansion and Transduction

Dynabeads CD8 Positive Isolation Kit (Life Technologies) was utilizedfor isolation of CD8 T cells from human PBMCs. Human CD8 T cells wereactivated with CD3/CD28 beads at a 1:1 ratio (Life Technologies) in acomplete RPMI media containing 40 IU/ml recombinant IL-2 for 72 hours.Activated T cells were re-suspended at concentration of 4 million cellsper 3 ml of FL1-CAR, FL2-CAR, FL3-CAR, CD19-CAR or Myc-Fc-CAR inlentiviral supernatant plus 1 ml of fresh RPMI media with 40 IU/ml IL-2and cultured in 6-well plates. Plates were centrifuged at 1200×g for 90minutes at 32° C. and then incubated for 4 hours at 37° C. Second andthird transductions were performed two more times.

Animal Experiments

All animal procedures were carried out in a strict accordance with therecommendations for proper use and care of laboratory animals (ECCDirective 86/609/EEC). The protocol was approved by the Inter-InstituteBioethics Commission of the Siberian Branch of the Russian Academy ofSciences (SB RAS). The experiments were conducted in the Center forGenetic Resources of Laboratory Animals at the Institute of Cytology andGenetics, SB RAS. Six- to eight-week-old female NOD SCID(CB17-Prkdc^(scid)/NciCrl) mice with an average weight of 16-20 g wereused. Tumors were engrafted by inoculating of 5×10⁶ Raji-FL1 cells in200 μL 0.9% saline solution subcutaneously into the left side of mice.Once tumors had reached a palpable volume of at least 50 mm³, mice wererandomly assigned to experimental or control groups. Tumor-bearing micewere injected intravenously (i.v.) with 3×10⁶FL1-CART, CD19-CART orMyc-CART cells on day 17^(th) post tumor inoculation. Tumor volume wasmeasured with calipers and estimated using the ellipsoidal formula.Animals were sacrificed when the volume of the tumor node reached 2 cm³.On the 38th day after tumor inoculation (21st day post CART infusion),animals from each experimental group were used for isolation of blood,spleen and bone marrow cells. Erythrocytes were lysed with RBC lysisbuffer (0.15 M NH₄Cl, 10 mM NaHCO₃, 0.1 mM EDTA) and cells were stainedwith antibodies specific for CD3 (for blood samples), CD45RA and CCR7and analyzed by Novocyte flow cytometer (ACEA Biosciences). The tumorswere fixed in 4% neutral buffered formaldehyde for 2 weeks and processedfor paraffin sectioning utilizing standard protocols.

Biophotonic Tumor Imaging

Animals were injected intraperitonealy with 150 μl (4.29 mg per mouse)of a freshly thawed aqueous solution of D-luciferin potassium salt(GOLDBIO). After 10 minutes animals were sacrificed and brain, lungs,heart, liver, spleen, kidneys, and tumors were collected. Each organ wasrinsed with PBS and bioluminescence intensity was visualized utilizingan In-Vivo MS FX PRO Imaging System (Carestream).

Histology and Immunohistochemistry

A macroscopic post-mortem analysis included examination of the externalsurfaces, appearance of primary tumor nodes, thoracic condition,abdominal and pelvic cavities with their associated organs and tissues.For further histological evaluation, specimens of tumor nodes from eachanimal were collected during autopsy and fixed in 10% neutral-bufferedformalin, dehydrated in ascending ethanols and xylols, and embedded inHISTOMIX paraffin (BioVitrum). Paraffin sections (5 μm) were stainedwith hematoxylin and eosin, microscopically examined and scanned. Tumorsections for immunohistochemical (IHC) studies (3-4 μm) were sliced on aMicrom HM 355S microtome (Thermo Fisher Scientific), and furtherde-paraffinated and rehydrated; antigen retrieval was carried out afterexposure in a microwave oven at 700 W. The samples were incubated withthe CD8-specific antibodies (M3164, Spring BioScience) according to themanufacture's protocol. Next, the sections were incubated with secondaryHRP-conjugated antibodies (Spring Bioscience detection system), exposedto DAB substrate, and stained with Mayer's hematoxylin. Images wereobtained using a Axiostar Plus microscope equipped with a Axiocam MRc5digital camera (Zeiss, Germany) at 10×, 20× and 40× magnifications.Gross examination of tumors included evaluation of size of the tumornode, presence of a capsule, and presence of necrosis and hemorrhages.Microscopic examination of tumors included evaluation ofhistopathological changes in tumor tissue in terms of necrosis andapoptosis, presence of mitoses and presence of CD8-lymphocyteinfiltration.

Statistics

The data obtained ex vivo (flow cytometry, cytotoxicity test) werestatistically processed using the Student's t-test (two-tailed,unpaired). The tumor volume measurements were statistically processedusing one-way ANOVA (STATISTICA 10.0). Survival curves were generatedusing the Kaplan-Meier method, and statistical comparisons wereperformed using the log-rank (Mantel-Cox) test. Significance wasconsidered for p≤0.05.

Cytotoxicity Assays

The cytotoxicity and specificity of engineered T cells were evaluated ina standard lactate dehydrogenase (LDH) release assay (CytoTox 96®Non-Radioactive Cytotoxicity Assay, Promega) following manufacturer'srecommendations. Mock transduced, CD19-CAR, FL1-CAR, FL2-CAR, FL3-CAR,or Myc-CAR T cells were co-incubated for 6 hours together with 104 ofthe Raji-FL1, Raji-FL2, Raji-FL3 or cells from the patient's biopsy in acomplete RPMI media supplemented with 40 U/ml of human IL-2. As negativecontrols Raji cells or cells isolated from an irrelevant lymphoma lymphnode biopsy were used. All the experiments were performed in triplicate.

Flow Cytometry Analysis

The following antibodies were used in this study; anti-human CD3 FITC(Biolegend), anti-human CD8 PE (Biolegend), anti-human CCR7 PE(Biolegend), anti-human CD45RA FITC (Biolegend), mouse anti-human CD69Alexa Fluor488 (Biolegend), anti-human B220 APC (Biolegend). ChimericFL-BCR expression was detected using anti-human IgG1 PE antibody(SouthernBiotech) or synthetic biotinylated cyclopeptides (GeneCust) andstreptavidin conjugated with FITC or PE (Thermo Fisher Scientific). TheCAR molecules were detected using goat cross-absorbed anti-human IgGantibody conjugated with DyLight650 (Thermo Fisher Scientific). TheCD19-CAR (FMC63 clone) molecules were detected using biotinylatedprotein L (Thermo Fisher Scientific) and streptavidin conjugated withFITC (Thermo Fisher Scientific).

Identification of the Bcl-2 Translocation

Crude DNA extracts were prepared by proteinase K digestion of follicularlymphoma lymph node biopsy sample. PCR amplification was carried outusing primer pairs comprising a consensus primer to JH and one of thethree different primers homological to sequences in the mbr1, mcr2 oricr5 regions of bcl2 gene as described in (14).

IFA

Self-reactivity of the lymphoma BCR was tested by indirectimmunofluorescence assay (IFA) on HEp-2 and HEL293T cells as describedin (15). Plasmid vector encoding recombinant myoferlin (22443, Addgene)was transfected into the HEK293T cells with Lipofectamine 2000(Invitrogen) as per the manufacturer's instructions. Recombinant Igsrepresenting lymphoma BCR and irrelevant human antibody were diluted inPBS with 2% BSA and used at a concentration of 50 μg/mL and incubatedwith cells for 1 hour. Detection of bound antibodies were accomplishedby anti-human Ig-PE using Nikon Eclipse Ti U microscope.

Results Overall Workflow

The aim of these proof-of-concept experiments is to find an antigen thatselectively reacts with the BCR on the surface of the lymphoma cell(FIG. 1). The central idea is that if the BCR can be cloned andexpressed on the surface of indicator cells also expressing a very largearray of peptides, the system becomes autocrine and each cell becomes aselection system onto itself. If the overall system is constructed suchthat the BCR signals when it reacts with one of the co-expressedligands, specific interactions between the BCR and the ligand can bereadily identified by FACS. Importantly, the autocrine-based selection,as used here, selects for functional interactions where antibody bindingto the peptide on the CAR activates the system.

Identification of the BCRs on Malignant B Cells

Lymph node biopsies from 3 patients with Follicular lymphoma (FL) wereused to determine the nucleotide sequence of the BCRs from malignantcells. The central part of the tumor biopsy was taken in order to reducethe abundance of BCR genes from non-malignant cells. Total mRNA was usedas a template in a reverse transcription reaction with subsequent PCRamplification of Ig V genes. Up to 95% percent of analyzed sequenceswere identical due to the clonal nature of lymphomas. The selected Igvariable regions were cloned into the pComb3X vector in a scFv format(S). Thus, the ScFv fused with constant domain of antibody (Fc) islinked via a flexible linker to a membrane-spanning domain of theplatelet-derived growth factor receptor (PDGFR) such that the antibodymolecules are integrated as dimers into the plasma membrane with theirbinding sites facing the solvent (S) (FIGS. 5B and 5C).

Autocrine-Based Selection of a Ligand for the BCR on the Malignant Cells

An autocrine-based reporter system for direct selection of ligands thatare specific to the BCR on malignant cells (FIG. 2A) was used. Themethod allows direct selection of a ligand that may be used for tumortargeting. T cells infected with both the BCR and combinatorialcyclopeptide library containing 10⁹ members were used as the reportersystem. Immortal Jurkat human T lymphocytes were modified tosimultaneously express the lymphoma BCR and a randomized 7 amino acidcyclopeptide library. The cyclopeptide library was fused with a chimericantigen receptor containing signaling domains (FIGS. 5A-5C). When the Igfused with the PDGFR membrane-spanning domain reacts with a peptide fromthe cyclopeptide library, the signaling domains of the chimericantigenic receptor trigger a T cell activation cascade. ActivatedT-cells start to express CD69 (early T-cell activation antigen) (6) andthus may be easily detected utilizing specific fluorescent-labeledantibodies.

First, the capacity of the reporter construction was confirmed using amodel system. A c-Myc epitope on CAR and the variable domains of theanti-Myc antibody (9E10 clone) was used as a model membrane bounded BCR.Jurkat cells expressing only membrane-bound anti-Myc antibody withoutco-expression of Myc-CAR showed no detectable activation. But, cellscontaining both membrane-bound anti-Myc antibody and Myc-CAR wereactivated FIG. 2B).

The results from the Myc model system encouraged us to move forward tothe actual BCR from the patient with lymphoma. In order to selectpeptide ligands of the reconstituted lymphoma BCRs, several rounds ofselection were performed, resulting in discovery of the threecyclopeptides CILDLPKFC (FL1) (SEQ ID NO: 1), CMPHWQNHC (FL2) (SEQ IDNO: 2) and CTTDQARKC (FL3) (SEQ ID NO: 3) specific for three patientderived BCRs scFv. Individual selected peptides-CAR fusions trigger a Tcell activation cascade in Jurkat cells when co-transduced bycorresponding membrane tethered BCRs as measured by CD69 membraneexpression (FIG. 2C).

Specific Lytic Activity Against Lymphoma Cells

Next, it was tested whether T cells transduced with the FL1-CAR, FL2-CARand FL3-CAR constructs demonstrated killing activity in vitro whenincubated with the Raji lymphoma cell lines transduced with the isolatedfollicular lymphoma B cell receptors (FL-BCR). Surface expression of thefunctional BCR from the malignant cells was confirmed by staining witha-Fc antibody and biotinylated FL1, FL2 and FL3 peptides (FIG. 3A).These studies confirmed that BCRs capable of binding to the peptideswere present on these cells.

To determine if CTLs expressing CAR-T were capable of killing targetcells, lentiviral vectors coding for the FL1-CAR, FL2-CAR, FL3-CAR orCD19-CAR were used to transduce human CD8⁺ T cells. Activated human CD8⁺T-cells baring peptide-CAR lysed Raji cells expressing the correspondingBCRs from the lymphomas (Raji-FL1, Raji-FL2 and Raji-FL3), as measuredby LDH release (FIG. 3B). Notably, the specific cytotoxicity of theFL1-CAR, FL2-CAR and FL3-CAR cells was comparable to the best-studiedCD19 CAR-T cell targeting CD19 antigen (FMC63-CAR). In contrast, minimumlysis was observed when control CARs T cells were used. Also, no celllysis was observed in case of incubation of FL1-CAR, FL2-CAR and FL3-CARwith unmodified Raji cells, suggesting high therapeutic potential andsafety of the BCR targeting CART (FIG. 6).

Next, cytotoxicity was estimated ex vivo of the FL1-CART against cellsfrom the patients 1 initial biopsy. More than 60% of cells in biopsysample are B-cells specific to the FL1 peptide (FIG. 3C, bottom panels).Cells from a control biopsy sample derived from another patient withfollicular lymphoma (patient 4) did not demonstrate any significantstaining by FL1 peptide. The CTL assay showed that FL1-CAR-Tspecifically lysed cells from the biopsy sample, while Myc-CAR-T andMock T cells did not have any anti-tumor lytic activity (FIG. 3D).

FL1-CAR Redirected CTLs Suppress Lymphoma Cells In Vivo

The efficacy of FL1-CART was tested in a relevant model of follicularlymphoma using immune-deficient NOD SCID (CB17-Prkdc^(scid)/NciCrl) miceengrafted with 5×10⁶ Raji cells expressing the FL1-BCR (Raji-FL1) (FIG.4A). Lentiviral vectors coding for FL1-CAR, Myc-CAR or CD19-CAR wereused to transduce CD3/CD28 bead-activated human CD8⁺ T cells resultingin a high efficiency of gene transfer (FIG. 4B). Injection of 5×10⁶FL1-CART or CD19-CART significantly suppressed the tumor burden andimproved survival in comparison with control group treated by Myc-CART(FIGS. 4C and 4D, FIGS. 7A-7C). On the 37th day 100% mice from thecontrol Myc-CART group were dead compared to 80% alive animals in theFL1-CART and CD19-CART groups. Flow cytometry was used to show thatCAR-modified T cells persist in peripheral blood 21 days post infusion,FL1-CAR-T and CD19-CAR-T cells were present in significantly elevatedamounts relative to Myc-CAR-T cells (FIG. 4D insert). As expected,expansion of CD8⁺ CAR-expressing T cells was correlated with expressionof surface markers associated with effector phenotypes (FIG. 4E).Interestingly, the population of FL1-CART in peripheral blood generallyconsisted of an effector memory subset, while spleen and bone marrowwere expanded by a central memory subset of cells (FIG. 4F). These latercells are thought to be important for persistence and sustainedanti-tumor activity.

Discussion

As immunotherapy expands, a way to discover more tumor antigens andtheir specific ligands is needed. At present the “menu” of tumorantigens is limited (7-12). However in the case of lymphomas the tumorantigen is already present as the BCR. Moreover, the BCR is an antibodywhose physiological role is to bind to antigen. This property of the BCRgreatly simplifies the problem of searching for ligands that interactwith the malignant BCR. Herein a “forced proximity” autocrine approach(13) was used, in which each reporter cell co-expresses one member of alarge peptide library on the cell surface together with the target BCRwhere they are co-integrated into the membranes of a population ofreporter cells. Several rounds of autocrine-based selection allowsdiscovery of a specific peptide ligand for the BCR.

It was demonstrated that T cells modified by these peptides fused withCAR efficiently eliminate tumor cells both, ex vivo and in vivo asefficiently as the well-known CD19-targeted CAR.

One advantage of this approach to antigen selection is that after therounds of panning the selected peptide ligands are already in aconstruct where they are fused to the chimeric antigen receptor. Thisallows one to immediately generate therapeutic T lymphocytes modified bytumor-specific CAR.

In essence the format reported here is the opposite of the usual CARTprotocol. Usually in cells bearing the CAR-T directionality is govern byantibody and target is a surface peptide or protein of the tumor cell.Here the inverse is used in that binding of the CAR-T is directed by thepeptide and a target is an antibody. Moreover, since the antibodymolecule is part of a huge diversity system, the target universe isbasically unlimited. This large target universe greatly simplifies theproblem of selecting ligands that are highly specific and tightlybinding.

As more patients are studied, the selected peptide sequences may be usedto determine the proteins they are derived from and by inference thedriving force for the malignant transformation. In this context, it isinteresting the discovered peptide is homologous to a region ofMyoferlin and identical to regions of surface proteins fromStreptococcus mitis and Pneumocytis jirovecii (FIGS. 8A-8E). Given thatthere is a suggestion that some lymphomas such as MALT are driven bysustained exposure to an infectious agent, the driving force forgeneration of lymphoid malignancies will be investigated as moreantigens that bind to the BCR are unearthed. Finally, the ability to usesequences other than CD19 as targets not only expands the choice in atherapeutic setting but also my help when the CD19 is absent or downregulated as may occur in many patients.

REFERENCES

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Example 2 Follicular Lymphoma

Lymphoma biopsy samples and patient mononuclear cell apheresis materialwere provided by N.N. Petrov Research Institute of Oncology (St.Petersburg, Russia) from a patient with advanced follicular lymphomascheduled to receive high dose chemotherapy and ASCT. CD34+ HSC wereisolated from apheresis material using anti-human CD34 microbeads andMACS cell separation technique as per the manufacturer's protocol(Miltenyi Biotech). Cell purity following MACS separation was >98% asdetermined by flow cytometry following staining of the purified cellswith anti-CD34-PE conjugated (Miltenyi). The remaining mononuclear cellfraction was used for isolation of CD8 T cells. Both CD34+ cells and CD8T cells were cryopreserved until use.

Fresh and viable samples of lymphoma tissue obtained through biopsy werecut at 3-5 mm³ pieces and implanted subcutaneously at multiple sites tofour six week old female NOD SCID (CB17-Prkdc^(scid)/NciCrl) (LaboratoryAnimals at the Institute of Cytology and Genetics, SB RAS).

Generation of Patient Specific Humanised Mice

Adult female NOD/SCID mice 5 weeks of age were acclimatized for at leasta 7-day period and were myeloablated by sublethal whole body irradiation(325 rad) delivered by a Gammacell 40 Exactor (Best Theratronics). 18mice were injected with 0.25×10⁶ purified CD34+ HSC cells per animal ina total volume of 200 mkl of phosphate-buffered saline (PBS) via thetail vein. All engrafted mice were housed under BL-2 conditions andprovided with autoclaved and water supplemented with Baytril(enrofloxacin).

Analysis of Immune Reconstitution of Patient Specific Humanised Mice

To measure the level of reconstitution with human immune cells followingstem cell transplant, mice were bled via the mandibular route (cheekpouch) using a sterile lancet (Braintree Scientific). Approximately ˜100mkl of blood was collected each time in K₂EDTA coated BD microtainercapillary blood collector tubes (Fisher Scientific).

The tubes were spun down at 500×g for 5 minutes for separation of theplasma. The cell pellet was treated with ACK lysis buffer to lyse RBCand washed extensively with MACS buffer containing BSA (Miltenyi) toenrich for peripheral blood mononuclear cells (PBMC).

Human PBMC, used as controls during flow cytometry analysis (FACS), waspurified from leukapheresis blood collars, following standard Ficolldensity gradient centrifugation techniques. Immunophenotyping wasperformed by staining the mononuclear cells with flurochrome conjugatedantibodies specific for different human immune cell surface markers(e.g., CD45, CD3, CD19, CD4, CD8, etc.) followed by multi-colour flowcytometry using a LSRII Flow Cytometr (Becton Dickinson, N.J.).Antibodies were obtained from eBioscience, Biolegend or BD Biosciences.During FACS, cell gating was done on viable lymphoid cells based on theforward and side scatter profile and most analysis performed on cellswithin the lymphoid gate.

A comparison between the percentages of human CD45+ and endogenous mouseCD45+ was performed to measure the level of immune reconstitution inmice. Background staining was determined using the corresponding isotypecontrols or staining cells isolated from unengrafted animals. Data wasanalyzed using the FlowJo software version 7.6.5 (Tree Star).

The electrochemiluminiscent based MSD platform (Meso Scale Discovery,Gaithersburg, Md.) was used to measure specific levels of human IgM andIgG in the plasma of mice at specific time points post transplantation.MSD 96-well High Bind Multi-Array plates were coated with 5 mkl ofeither anti-human IgM or anti-human IgG Fc (Bethyl Laboratories) at aconcentration of 20 mkg/ml per well at 4° C. overnight. Plates wereblocked with PBS/2% fetal bovine serum for 1 h followed by repeatedwashing with PBS/0.05% Tween-20. Mouse plasma samples were tested at1:50-1:100 dilutions for human IgG levels and 1:500-1:1000 dilutions forhuman IgM levels in a total volume of 20 μl for each sample added perwell in duplicates. Following incubation and washing as describedearlier, 20 μl goat anti-human Ig antibody with SULFO-Tag at aconcentration of 2 μg/ml per well was used as the detection Ab andplates incubated for 1 h at room temperature. Plates were developed byadding the appropriate substrate and read on the MSD Sector Imager 2400according to the manufacturer's protocol. Human IgM and IgG standards(Bethyl Labs) was used to obtain the standard curve and human of Iglevels computed using GraphPad Prism program version 5. The results aresummarized in FIGS. 9-11.

Identification of the BCR on the malignant B cell is specified in RU2017134483. Autocrine-based selection of a ligand for the BCR on themalignant cells is specified in RU 2017134483. Lentiviral CAR Tconstruct is specified in RU 2017134483.

CD8⁺ T Cell Activation, Expansion and Transduction

Dynabeads CD8 Positive Isolation Kit (Life Technologies) was utilizedfor isolation of CD8 T cells from patient PBMCs fraction collected byapheresis. Human CD8 T cells were activated with CD3/CD28 beads at a 1:1ratio (Life Technologies) in a complete RPMI media containing 40 IU/mlrecombinant IL-2 for 72 hours. Activated T cells were re-suspended atconcentration of 4 million cells per 3 ml of FL1-CART in lentiviralsupernatant plus 1 ml of fresh RPMI media with 40 IU/ml IL-2 andcultured in 6-well plates. Plates were centrifuged at 1200×g for 90minutes at 32° C. and then incubated for 4 hours at 37° C. Second andthird transductions were performed two more times.

BCR Vaccination

In order to obtain the soluble form of patient follicular lymphoma BCRas a full-size antibody, VH and VL were cloned into the pFUSE antibodyexpression vectors (Invivogen) and produced utilizing FreeStyle 293Expression System (Thermo Fisher Scientific). Protein was furtherpurified and coupled to keyhole limpet hemocyanin using 0.1%glutaraldehyde as described by Levy (R. Levy. 1987 et al., Idiotypevaccination against murine B cell lymphoma. Humoral and cellularresponses elicited by tumor-derived IgM and its molecular subunits. JImmunol. 139:2825). Human IgG Isotype Control antibody (Invitrogen, cat12000C) was conjugated to keyhole limpet hemocyanin as used as thecontrol vaccine. Mice were immunized using subcutaneous injections with0.1 ml with an emulsion of equal parts Freund's complete adjuvant andKLH-IgG at 100 mkg/ml in PBS.

Animal Experiments

All animal procedures were carried out in a strict accordance with therecommendations for proper use and care of laboratory animals (ECCDirective 86/609/EEC. All mouse surgical procedures and imaging wereperformed with the animals anesthetized by intramuscular injection of a0.02 ml solution of 50% ketamine, 38% xylazine, and 12% acepromazinemaleate. Patient B Cell FL tumor nodules were excised from female NODSCID (CB17-Prkdc^(scid)/NcCrl) mice, tumor fragments without evidence ofnecrosis were sliced to equal 3 mm³ pieces and transplantedsubcutaneously to sixteen NOD/SCID mice with reconstituted patientimmune system at 18 w age. Tumor volume was measured with calipers andestimated using the formula a/6×(lengt ×width×height). Mice were dividedinto three experimental groups treated as follows:

-   -   Group 1: 3×10⁶ FL1-CART intravenously at day 10 after transplant    -   Group 2: KLH-patient BCR vaccine subcutaneously at days 1, 5, 15        after transplant    -   Group 3: 3×10⁶ FL1-CART intravenously at day 10 after        transplant+KLH− patient BCR vaccine subcutaneously at days 1, 5        and 15 after transplant    -   Group 4: 3×10⁶ FL1-CART intravenously at day 10 after        transplant+KLH isotype control vaccine subcutaneously at days 1,        5 and 15 after transplant Animals were sacrificed at day 38        following transplant. Tumor growth kinetics in experimental        groups are presented in FIG. 12.

Thus, the combination of intravenous FL1-CART therapy and vaccinationusing the patient BCR vaccine results in synergistic suppression oftumor growth. Opposite to that, the combination of intravenous FL1-CARTtherapy with isotype control vaccine reduces efficacy of FL1-CARTtherapy.

Flow Cytometry Analysis

On the 38th day after tumor inoculation (21st day post CART infusion),animals from each experimental group were used for isolation of blood.Erythrocytes were lysed with RBC lysis buffer (0.15 M NH₄Cl, 10 mMNaHCO₃, 0.1 mM EDTA). Chimeric FL-BCR expression was detected usingsynthetic ACILDLPKFCGGGS-Bio (SEQ ID NO: 29) cyclopeptide (GeneCust) andstreptavidin conjugated with FITC (Thermo Fisher Scientific) andanalyzed by Novocyte flow cytometer (ACEA Biosciences).

The combination of intravenous FL1-CART therapy and vaccination usingpatient BCR vaccine results in the highest levels of FL1-CART cells incirculation while concomitant vaccination with isotype control vaccinedo not produce any synergy.

Meso-Scale Based Analysis of Specific Antibody Responses:

MSD analysis of the terminal plasma samples were performed to measureantibody responses against the patient specific BCR and IgG IsotypeControl

Patient BCR antigen and Isotype Control antigens were coated on highbind MSD 96-well plates at concentrations between 20-50 mkg/ml with 5mkl added per well and incubated overnight at 4° C. Plasma samples weretested at 1:80 dilution. Sulfo-tagged Anti-human Ig was used as thedetection antibody and reaction developed using anelectrochemiluminiscent (ECL) substrate and read in a MSD Sector Imager2400 (Meso Scale Discovery).

TABLE 2 Anti BCR and Isotype Control antibody responses (MSD relativeunits) in immunized mice on day 38. Group 1 Group 2 Group 3 Group 4 BCR700 ± 115 4700 ± 610  9700 ± 1550 1150 ± 175 IgG Isotype Control 690 ±225 950 ± 170 1050 ± 135  3950 ± 375 MSD analysis of plasma reactivityto the respective antigens were measured and compared.Data is represented as mean+/−SEM. The combination of intravenousFL1-CART therapy and vaccination using patient BCR vaccine results inthe highest levels of anti BCR reactivity versus BCR vaccine alone.

The data above clearly confirm the finding of substantial therapeuticsynergy (tumor growth inhibition and level of personalized cancerantigen directed CAR T cells and immunoglobulins) between CAR T adoptiveimmunotherapy and vaccination wherein both targets same personalizedcancer antigen.

NSCLC Harbouring EGFRvII Mutation

To further confirm the observations, a patient with advanced NSCLC whowas scheduled to undergo a cytoreductive surgery at Advanced SurgeryDepartment of Kirov Academy of Military Medicine (St. Petersburg) andwhose tumor tissue was positive for EGFRvIII mutation as confirmed byICH staining of biopsy material was identified.

Epidermal growth factor receptor variant III (EGFRvIII) is the result ofa novel tumor-specific gene rearrangement that produces a unique proteinexpressed in approximately 30% of gliomas, and certain other cancersincluding lung, breast and ovarian cancers. By deletion of a segment ofthe ligand-binding domain, EGFRvIII bypasses the need of ligand. Thisdeletion spans exons 2-7, resulting in the introduction of a novelglycine residue at the fusion junction. While this mutant cannot bindligands, it resides at the cell membrane and present a case ofwell-established personalized cancer model antigen harbouring a tumorspecific mutation.

Two weeks prior to surgery patients were mobilized with 10 mkg/kg ofGM-CSF (Neostim, Pharmsynthez) administered subcutaneously once a dayfor 5 consecutive days. Mobilized peripheral blood stem cells werecollected on the Cobe Spectra Apheresis system. Approximately 3-6 bloodvolumes were processed during each daily collection, which lasted up to11 hours. Patient underwent two daily apheresis procedures to collect2×10⁶ CD34+ cells per kg. CD34+ HSC were isolated from apheresismaterial using anti-human CD34 microbeads and MACS cell separationtechnique as per the manufacturer's protocol (Miltenyi Biotech). Cellpurity following MACS separation was >98% as determined by flowcytometry following staining of the purified cells with anti-CD34-PEconjugated (Miltenyi). The remaining mononuclear cell fraction was usedfor isolation of CD8 T cells. Both CD34+ cells and CD8 T cells werecryopreserved until use. Fresh and viable samples of tumor tissueobtained during patient surgery were cut at 3-5 mm³ pieces and implantedsubcutaneously at multiple sites to four six week old female NOD SCID(CB17-Prkdc^(scid)/NcrCrl) (Laboratory Animals at the Institute ofCytology and Genetics, SB RAS).

Generation of Patient Specific Humanised Mice

Adult female NOD/SCID mice 5 weeks of age were acclimatized for at leasta 7-day period and were myeloablated by sublethal whole body irradiation(325 rad) delivered by a Gammacell 40 Exactor (Best Theratronics). 18mice were injected with 0.25×10⁶ purified CD34+ HSC cells per animal ina total volume of 200 mkl of phosphate-buffered saline (PBS) via thetail vein. All engrafted mice were housed under BL-2 conditions andprovided with autoclaved and water supplemented with Baytril(enrofloxacin).

Analysis of Immune Reconstitution

Analysis of immune reconstitution was performed as described above at 6,12 and 18 wk. Data is presented in FIGS. 14-16.

CAR T Lentiviral Vector

A EGFRvIII targeting 139-scFv-based CAR vector was assembled using scFvsequence from human anti-EGFRvIII antibody 131 to T-cell signallingdomains from CD28-41BB-CD3(as described by Rosenberg (Steven A.Rosenberg et al., Recognition of Glioma Stem Cells by GeneticallyModified T Cells Targeting EGFRvIII and Development of Adoptive CellTherapy for Glioma, Hum Gene Ther. 2012 October; 23(10): 1043-1053.) DNAfragment coding for CD28-41BB-CD3ζ was synthesized (GeneCust) and clonedinto the pLV2 lentiviral vector (Clontech) under control of the EF1apromoter. The arrangement of genes is in the order of: IL2-signalsequence, 139-scFv, GGGS linker; a CD28 trans-membrane and intracellularregion; intracellular domains of the OX-40 and CD3zetta. Thelentiviruses were prepared by co-transfection of HEK293T cells with thepLV2-139-scFv-CD28-41BB-CD3zetta plasmid and the packaging plasmids(2^(nd) generation). Supernatants containing the virus were collected at48 h post transfection. The titer of lentivirus preparations wasdetermined using Lenti-X p24 ELISAs (Clontech).

CD8⁺ T Cell Activation, Expansion and Transduction

Dynabeads CD8 Positive Isolation Kit (Life Technologies) was utilizedfor isolation of CD8 T cells from patient PBMCs fraction collected byapheresis. Human CD8 T cells were activated with CD3/CD28 beads at a 1:1ratio (Life Technologies) in a complete RPMI media containing 40 IU/mlrecombinant IL-2 for 72 hours. Activated T cells were re-suspended atconcentration of 4 million cells per 3 ml of CD28-41BB-CD3ζ-CAR inlentiviral supernatant plus 1 ml of fresh RPMI media with 40 IU/ml IL-2and cultured in 6-well plates. Plates were centrifuged at 1200×g for 90minutes at 32° C. and then incubated for 4 hours at 37° C. Second andthird transductions were performed two more times.

Vaccination

14-amino acid peptide corresponding to the amino acid sequence at thefusion junction (LEEKKGNYVVTDHC) (SEQ ID NO: 30), was synthesized,purified, and coupled to keyhole limpet hemocyanin as described byBigner (Monoclonal Antibodies against EGFRvIII are Tumor Specific andReact with Breast and Lung Carcinomas and Malignant Gliomas. Darell D.Bigner et al., Cancer Res. 1995 Jul. 15; 55(14):3140-8). LEEKKGNYVVTDHC(SEQ ID NO: 30) is an epitope recognized by antibody 139, used forengineering of 139-scFv-based CAR. Mice were immunized usingsubcutaneous injections with 0.1 ml with an emulsion of equal partsFreund's complete adjuvant and KLH-LEEK at 100 mkg/ml in PBS.

Animal Experiments

All animal procedures were carried out in a strict accordance with therecommendations for proper use and care of laboratory animals (ECCDirective 86/609/EEC. All mouse surgical procedures and imaging wereperformed with the animals anesthetized by intramuscular injection of a0.02 ml solution of 50% ketamine, 38% xylazine, and 12% acepromazinemaleate. Patient NSCLC tumor nodules were excised from female NOD SCID(CB17-Prkdc^(scid)/NciCrl) mice, tumor fragments without evidence ofnecrosis were sliced to equal 3 mm³ pieces and transplantedsubcutaneously to fifteen NOD/SCID mice with reconstituted patientimmune system at 18 w age. Tumor volume was measured with calipers andestimated using the formula π/6×(length×width×height). Mice were dividedinto three experimental groups treated as follows:

-   -   Group 1: 3×10⁶ CD28-41BB-CD3ζ-CART intravenously at day 10 after        transplant    -   Group 2: KLH-LEEK vaccine subcutaneously at days 1,5,15 after        transplant    -   Group 3: 3×10⁶ CD28-41BB-CD3ζ-CART intravenously at day 10 after        transplant+KLH-LEEK vaccine subcutaneously at days 1, 5 and 15        after transplant        Animals were sacrificed at day 38 following transplant. Tumor        growth kinetics in experimental groups is presented in FIG. 18.

‘The data confirm the finding of substantial therapeutic synergy betweenCAR T adoptive immunotherapy and vaccination wherein both targets samepersonalized cancer antigen.

Example 3 Method for Identification of B Cell Receptor Ligand by PhageDisplay

As a general alternative to the Reporter Cells a phage-displayedcyclopeptide library panning may be performed for identification of themalignant BCR specific moiety. Commercially available phage-peptidelibraries such as New England Biolabs Ph.D.-7 and Ph.D.-12 libraries maybe utilized. For randomization Ph.D.™-C7C Phage Display CyclopeptideLibrary Kit uses NNK coding moiety flanked by Cysteines shown in FIG.19. Herein, we provide modified NEB protocol for a malignant BCRspecific peptides identification. It is recommended to performnegative-selection incubation during each round of panning.

Panning Procedure:

-   -   1. Inoculate 10 ml of LB+Tet medium with ER2738, for use in        titering. If amplifying the eluted phage on the same day, also        inoculate 20 ml of LB medium in a 250-ml Erlenmeyer flask (do        not use a 50-ml conical tube) with ER2738. Incubate both        cultures at 37° C. with vigorous shaking. ER2738 is E. coli host        strain F′ proA+B+ lacIq Δ(lacZ)M15 zzf::Tn10(TetR)/fhuA2 glnV        Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5.    -   2. Transfer 50 μl of a 50% aqueous suspension of affinity beads        appropriate for capture of the antibody to a microfuge tube. Add        1 ml of TBS+0.1% Tween (TBST). Suspend the resin by tapping the        tube.    -   3. Pellet the resin by magnetic capture. Carefully pipette away        and discard the supernatant.    -   4. Suspend the resin in 1 ml of blocking buffer (0.1 M NaHCO₃(pH        8.6), 5 mg/ml BSA, 0.02% NaN3 (optional). Filter sterilize,        store at 4° C.).    -   5. Incubate for 60 minutes at 4C, mixing occasionally.    -   6. In the meantime, mix the 2×10⁹ phages with 2 mkg of a        negative-selection antibody to a final volume of 200 μl with        TBST.    -   7. Incubate for 20 minutes at room temperature.    -   8. Following the blocking reaction in Step 4, pellet the resin        as in Step 3 and wash 4 times with 1 ml of TBST, pelleting the        resin each time.    -   9. Resuspend resing in 1 ml and aliquote to a two separate tubes        (500 mkl each).    -   10. Transfer the phage-neg-antibody mixture to the first tube        containing the washed resin. Mix gently and incubate for 15        minutes at room temperature, mixing occasionally.    -   11. Pellet the resin as in Step 3, collect the supernatant.    -   12. Mix the supernatant with 2 mkg of the malignant BCR        antibody.    -   13. Incubate for 20 minutes at room temperature.    -   14. Pellet the resin as in Step 3, discard the supernatant, and        wash 10 times with 1 ml of TBST, pelleting the resin each time.    -   15. Elute the bound phage by suspending the resin in 1 ml of        Glycine Elution Buffer (0.2 M Glycine-HCl, pH 2.2, 1 mg/ml BSA).    -   16. Incubate for 10 minutes at room temperature.    -   17. Pellet resin by magnetization for 1 minute.    -   18. Carefully transfer the supernatant to a new microfuge tube,        taking care not to disturb the pelleted resin.    -   19. Immediately neutralize the eluate with 150 μl of 1 M        Tris-HCl, pH 9.1.    -   20. Amplify the remaining eluate by adding it to the 20 ml        ER2738 culture from Step 1 (must be early-log; no later) and        incubating at 37° C. with vigorous shaking for 4.5 hours.        -   21. Transfer the culture to a centrifuge tube and spin for            10 minutes at 12,000 g at 4° C. Transfer the supernatant to            a fresh tube and re-spin (discard the pellet).    -   22. Pipette the upper 80% of the supernatant to a fresh tube and        add to it 1/6 volume of 20% PEG/2.5 M NaCl.    -   23. Allow the phage to precipitate at 4° C. for 2 hours or        overnight.    -   24. Spin the PEG precipitation at 12,000 g rpm for 15 minutes at        4° C.    -   25. Decant and discard the supernatant, respin briefly, and        remove the residual supernatant with a pipette.    -   26. Suspend the pellet in 1 ml of TBS. Transfer the suspension        to a tube and spin for 5 minutes at 4° C. to pellet residual        cells.    -   27. Transfer the supernatant to a fresh microcentrifuge tube and        reprecipitate with 1/6 volume of 20% PEG/2.5 M NaCl.    -   28. Incubate for 15-60 minutes on ice.    -   29. Microcentrifuge at 14,000 rpm for 10 minutes at 4° C.    -   30. Discard the supernatant, respin briefly, and remove residual        supernatant with a micropipet.    -   31. Suspend the pellet in 200 μl of TBS.    -   32. Microcentrifuge at 14,000 rpm for 1 minute to pellet any        remaining insoluble matter.    -   33. Transfer the supernatant to a fresh tube. This is the        amplified eluate.    -   34. Perform a second and third rounds of panning.

Plaque Amplification for ELISA and Sequencing:

-   -   1. Dilute an overnight culture of ER2738 1:100 in LB. Dispense 1        ml of diluted culture into 96-well deepwell plates (#260251,        Thermo Scientific). For each antibody to be characterized use 2        plates.    -   2. Stab a blue plaque from a phage plates.    -   3. Use a microplate tape sealer to cover the plates.    -   4. Incubate the plates at 37° C. with shaking for 4.5-5 hours.    -   5. Centrifuge plates at 250 g for 10 minutes at RT.    -   6. Carefully collect 700 mkl of the supenatant and transfer to a        fresh plate.    -   7. This is the amplified phage stock and can be stored at 4C for        two days.

Phage ELISA Binding Assay:

-   -   1. Coat ELISA plate wells with 100 μl of 100 μg/ml of malignant        BCR antibody or negative-control antibody in 0.1 M NaHCO₃, pH        8.6.    -   2. Incubate overnight at 4C.    -   3. Wash each plate 5 times with TBST.    -   4. Block ELISA plate wells by 5% Milk in PBST.    -   5. Incubate 1 hour at RT.    -   6. Wash each plate 5 times with TBST.    -   7. In the separate blocked plate, carry out fourfold serial        dilutions of the phage supernantant.    -   8. Using a multichannel pipettor, transfer 100 μl from each row        of diluted phage to a row of antibody-coated wells.    -   9. Incubate at RT for 2 hours with agitation.    -   10. Wash each plate 5 times with TBST.    -   11. Dilute HRP-conjugated anti-M13 monoclonal antibody (GE        Healthcare. #27-9421-01) in blocking buffer to the final        dilution recommended by the manufacturer. Add 200 μl of diluted        conjugate to each well.    -   12. Incubate at RT for 1 hour with agitation.    -   13. Add 50 μl of substrate solution to each well, and incubate        for 10-60 minutes at room temperature with gentle agitation.    -   14. Read the plates using a microplate reader set at 415 nm. For        each phage clone, compare the signals obtained with        negative-control and malignant BCR antibody.

Sequencing of Phage DNA:

-   -   1. Transfer 500 μl of the phage-containing supernatant to a        fresh microfuge tube.    -   2. Add 200 μl of 20% PEG/2.5 M NaCl. Invert several times to        mix, and let stand for 10-20 minutes at room temperature.    -   3. Microfuge at 14,000 rpm for 10 minutes at 4C and discard the        supernatant. Phage pellet may not be visible.    -   4. Re-spin briefly. Carefully pipet away and discard any        remaining supernatant.    -   5. Suspend the pellet thoroughly in 100 μl of Iodide Buffer by        vigorously tapping the tube.    -   6. Add 250 μl of ethanol.    -   7. Incubate 10-20 minutes at room temperature.    -   8. Spin in a microfuge at 14,000 rpm for 10 minutes at 4C.    -   9. Discard the supernatant.    -   10. Wash the pellet with 0.5 ml of ice-cold 70% ethanol.    -   11. Suspend the pellet in 30 μl of TE buffer.    -   12. Use 5 μl of the DNA in TE buffer as a template for        sequencing.    -   13. Use the reverse primer for DNA sequencing (GCA ATG CGA TTG        ATA CTC CC (SEQ ID NO: 41)).

Results of the panning are shown in the following tables. For thedisplay the full-size follicular lymphoma BCR in IgG1 format was used.Patent FL1 is as described in Example 1. Table 3 shows ELISA results forthe binding of amplified phages resulting from I-III rounds of panningagainst the BCR of patient FL1 with the BCR of patients FL1 and FL5 atthe phage concentrations shown. Results are also shown in FIG. 20.

FL1 Ab FL5 Ab Phages amount FL1 Ab phage rounds FL1 Ab phage roundsmkl/well 1 2 3 1 2 3 5 0.1234 2.1417 2.6927 0.2405 0.2538 0.2753 2.500.0656 0.873 2.5545 0.122 0.1241 0.133 1.25 0.0577 0.2054 1.5719 0.0930.1014 0.1127 0.63 0.0534 0.0868 0.5184 0.092 0.0948 0.0982 0.31 0.05230.056 0.1392 0.091 0.0901 0.1Table 4 shows ELISA results for the binding of phages from individualplaques after III rounds of panning against the BCR of patient FL1 withthe BCR of patient FL1.

1 2 3 4 5 6 7 8 9 10 11 12 A 0.063 2.024 1.313 1.305 1.132 0.683 0.9080.718 0.142 0.379 0.225 0.098 B 0.054 0.053 0.117 0.073 1.013 0.0681.135 0.436 0.1 0.062 0.537 0.695 C 1.015 0.054 0.495 0.743 0.639 0.4560.076 0.213 0.061 0.728 0.626 0.639 D 0.055 0.783 0.808 0.522 0.754 0.430.513 0.443 0.499 0.27 0.147 0.519 E 1.444 0.682 0.564 0.592 0.08 0.420.519 0.088 0.111 0.368 0.316 0.055 F 0.871 0.371 0.627 0.604 0.4910.159 0.371 0.128 0.316 0.241 0.12 0.647 G 0.794 0.909 0.573 0.453 0.4840.435 0.136 0.379 0.598 0.517 0.525 0.501 H 0.079 1.229 0.205 0.4151.459 0.36 0.231 0.12 0.075 0.522 0.409 0.091Table 5 shows ELISA results for the binding of phages from individualplaques after III rounds of panning against the BCR of patient FL1 withthe BCR of patient FL5.

1 2 3 4 5 6 7 8 9 10 11 12 A 0.081 0.076 0.056 0.074 0.108 0.073 0.0880.124 0.088 0.063 0.064 0.074 B 0.059 0.066 0.077 0.102 0.117 0.0630.079 0.067 0.097 0.062 0.093 0.211 C 0.121 0.085 0.091 0.161 0.0740.091 0.058 0.091 0.059 0.068 0.093 0.204 D 0.073 0.123 0.097 0.0880.098 0.101 0.083 0.08 0.071 0.066 0.064 0.196 E 0.067 0.134 0.101 0.0680.067 0.086 0.092 0.067 0.058 0.141 0.095 0.061 F 0.082 0.276 0.1180.109 0.064 0.08 0.059 0.059 0.065 0.118 0.068 0.092 G 0.179 0.188 0.1060.129 0.087 0.143 0.063 0.106 0.108 0.106 0.227 0.127 H 0.102 0.0830.118 0.15 0.133 0.076 0.08 0.13 0.06 0.119 0.117 0.149

The positive clones from Table 4 were amplified and sequenced. Thesequence, location on Table 4, and OD are shown below in Table 6. Thepeptide identified as binding the BCR of patient FL1 was also identifiedas a BCR ligand in Example 1 using the autocrine signaling method.

TABLE 6 Sequence (SEQ ID NO: 42) Position OD  1 ILDLPKF C1 1.02  2ILDLPKF E1 1.44  3 ILDLPKF A2 2.02  4 ILDLPKF H2 1.23  5 ILDLPKF A3 1.31 6 ILDLPKF A4 1.31  7 ILDLPKF A5 1.13  8 ILDLPKF B5 1.01  9 ILDLPKF H51.46 10 ILDLPKF B7 1.14

After the cyclopeptide specific for the FL1 patient's BCR wasidentified, as shown in Table 6, the sequence was cloned into a3-generation CAR lentiviral vector. Two complementary primers coding forthe selected cyclopeptide flaked by EcoRI and NheI cloning sites weresynthesized.

Primer FL1peptide FW (SEQ ID NO: 43) TCACGAATTCGGCTTGTATTCTTGATTTGCCGAAGTTTTGCGGTGGAGGT TCGGCTAGCPrimer FL1peptide Rev (SEQ ID NO: 44)GCTCGCTAGCCGAACCTCCACCGCAAAACTTCGGCAAATCAAGAATACA

After amplification the PCR product was cloned into the pLV2-Fc-CARvector at the EcoRI and NheI restriction sites.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

What is claimed is:
 1. A method of treating cancer in a subjectcomprising concomitantly administering: CAR-expressing T-cells, whereinthe CAR comprises an antigen binding domain that specifically binds acancer-specific antigen in a cancer-specific manner; and a vaccinecomprising a polypeptide or a nucleic acid expressing thecancer-specific antigen, or a cancer-specific fragment thereof.
 2. Themethod of claim 1, wherein the cancer-specific antigen is a B-cellreceptor.
 3. The method of claim 1, wherein the polypeptide or nucleicacid comprises a heavy or light chain variable region, or fragmentthereof.
 4. The method of claim 1, wherein the cancer-specific antigenis expressed in the cancer and comprises a somatic mutation.
 5. Themethod of claim 1, wherein the cancer-specific antigen is expressed inthe cancer and comprises a somatic mutation.
 6. The method of claim 5,wherein the non-cancerous cells of the subject do not have the somaticmutation.
 7. The method of claim 5, wherein the mutation is a pointmutation, a splice-site mutation, a frameshift mutation, a read-throughmutation, or a gene-fusion mutation.
 8. The method of claim 5, whereinthe somatic mutation comprises a mutation in EGFRvIII, PSCA, BCMA, CD30,CEA, CD22, L1CAM, ROR1, ErbB, CD123, IL13Rα2, Mesothelin, FRα, VEGFR,c-Met, 5T4, CD44v6, B7-H4, CD133, CD138, CD33, CD28, GPC3, EphA2, CD19,ACVR2B, anaplastic lymphoma kinase (ALK), MYCN, BCR, HER2, NY-ESO1,MUC1, or MUC16.
 9. The method of claim 5, wherein the cancer comprises atumor.
 10. The method of claim 5, wherein the polypeptide or nucleicacid comprises the somatic mutation.
 11. The method of claim 5, whereinthe concomitant administration occurs at least two times, at least threetimes, at least four times, at least five times, at least six times, atleast seven times, at least eight times, at least nine times, or atleast ten times in the subject.
 12. The method of claim 5, wherein theCAR-expressing T-cells are administered before the vaccine.
 13. Themethod of claim 5, wherein the CAR-expressing T-cells are administeredafter the vaccine.
 14. The method of claim 5, further comprisingidentifying the cancer-specific antigen in the subject.
 15. The methodof claim 14, wherein identifying the cancer-specific antigen comprises:(i) obtaining cancerous cells from a subject; (ii) extracting DNA fromthe cells; and (iii) sequencing the DNA.
 16. The method of claim 15,wherein identifying the cancer-specific antigen further comprisescomparing the DNA sequence obtained from the cancerous cells to a DNAsequence of the same gene obtained from non-cancerous cells.
 17. Themethod of claim 14, wherein the DNA is isolated from tumor cells. 18.The method of claim 14, wherein identifying the cancer-specific antigencomprises isolating and sequencing circulating cell free DNA of thesubject.
 19. The method of claim 14, wherein identifying thecancer-specific antigen comprises: (i) obtaining cancerous cells from asubject; (ii) extracting RNA from the cells; (iii) synthesizing cDNAfrom the extracted RNA; and (iv) sequencing the cDNA.
 20. The method ofclaim 19, wherein identifying the cancer-specific antigen furthercomprises comparing the cDNA sequence obtained from the cancerous cellsto a cDNA sequence of the same gene obtained from non-cancerous cells.21. The method of claim 5, wherein the vaccine comprises two or morepolypeptides having overlapping sequences, each expressing a fragment ofthe cancer-specific antigen.
 22. The method of claim 5, furthercomprising providing CAR-expressing T-cells by: (i) identifying anantigen binding domain that specifically binds the cancer-specificantigen in a cancer-specific manner; and (ii) expressing a CARcomprising the antigen binding domain in T-cells.
 23. The method ofclaim 5, wherein the CAR comprises a transmembrane domain that comprisesalpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon,CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86,CD134, CD137 and/or CD154.
 24. The method of claim 5, wherein the CARcomprises an intracellular region.
 25. The method of claim 24, whereinthe intracellular region comprises a MHC class I molecule, a TNFreceptor protein, an Immunoglobulin-like protein, a cytokine receptor,an integrin, a signaling lymphocytic activation molecule (SLAM protein),an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2,CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD25/CD18), 4-29B(CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1,CD423, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE,CD103, ITGAL, CD11a, LFA-1, ITGAM, CD129, ITGAX, CD11c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4 (CD244, 304), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD123, a ligand that specificallybinds with CD83, and/or CD3 zeta.